CN107011443A - For the composition for the complement activation for suppressing the dependences of MASP 2 - Google Patents

Abstract

The present invention relates to the anti-inhibiting antibodies of MASP 2 and the composition including the antibody, the ill effect of the complement activation for suppressing the dependences of MASP 2.On the one hand, the invention provides the human monoclonal antibodies of separation or its antigen-binding fragment, it combines people MASP 2, and the antibody includes：（i）Weight chain variable district including CDR H1, CDR H2 and CDR H3 sequences；With（ii）Light chain variable district including CDR L1, CDR L2 and CDR L3 sequences, wherein the weight chain variable district CDR H3 sequences include setting forms extra alternative route C3 convertase with Factor B（C3bBb）Amino acid sequence.Alternative route C3 convertase is stabilized by combining properdin.Properdin makes six to ten times of the Increased Plasma Half-life of alternative route C3 convertase.Cause the formation of alternative route C5 convertase to alternative route C3 convertase addition C3b.

Description

For the composition for the complement activation for suppressing MASP-2 dependences

The application is divisional application, and the Chinese application number of its female case is：201280032866.X, international application no is PCT/ US2012/036509, the applying date is on May 4th, 2012.

Technical field

The present invention relates to anti-MASP-2 inhibiting antibodies and the composition including the antibody, for suppressing MASP-2 dependences The ill effect of complement activation.

The cross reference of related application

The application asks the rights and interests for the U.S. Provisional Application No. 61/482,567 submitted on May 4th, 2011, and it is overall by quoting It is incorporated herein.

Statement on sequence table

The related sequence table of the application is provided with text formatting replaces paper copy, and accordingly by being incorporated by specification. The title of text comprising sequence table is MP_1_0115_PCT_SequenceListingasFiled_20120504_ ST25.Text is 158KB, is founded on May 4th, 2012, and just carried together with the submission of specification via EFS-Web Hand over.

Background technology

Complement system is to start, strengthen and coordinate in people and other vertebrates to microorganism infection and other acute injuries Immune response provides the mechanism of action of early stage, and (M.K. Liszewski and J.P. Atkinson, 1993, are loaded inFundamental Immunology, the third edition, W.E. Paul edit, Raven Press, Ltd., New York).Although mending Body activation provides the important First Line for potential pathogen and defendd, but promotes the complement activity of protective immune response Potential threat (K.R. Kalli etc., the Springer Semin. to host can also be shownImmunopathol. 15: 417-431, 1994；B.P. Morgan,Eur. J. Clinical Investig.24:219-228, 1994).For example, C3 and C5 protein hydrolysates are raised and activate neutrophil cell.Although being essential for host defense, live The neutrophil cell of change is indiscriminate in the release of their destructive enzymes, it is possible to cause organ damage.In addition, mending Body activation can cause the complement component of lysis to deposit on nigh host cell and microorganism target, cause host cell to split Solution.

The pathogenesis implication of complement system with many acute and chronic morbid states, the disease includes：The heart Muscle infarction, apoplexy, ARDS（ARDS）, reperfusion injury, septic shock, the capillary after thermal burn The postoperative inflammation of vascular leakage, cardiovascular shunt, graft rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis and A Er Ci Haimo diseases.In these almost all of situations, complement is not the cause of disease, but a number of factors involved by pathogenesis it One.Nevertheless, complement activation can be important pathomechanism, and the clinic control in many this kind of morbid states is showed Go out effective part.

Gradually increased understanding to the importance of the tissue damage of complement-mediated in various morbid states is strengthened to having Imitate the demand of Complement inhibition medicine.To current, Yi Kuli monoclonal antibodies（Soliris®）, it is only to be criticized for C5 antibody The mutatis mutandis complement targeted drug used in people.However, C5 is positioned at one of several effector molecules of complement system " downstream ", and And blocking C5 does not suppress the activation of complement system.Therefore, the inhibitor of the initial step of complement activation will have in relative " downstream " The significant advantage of complement inhibitor.

At present, generally receiving complement system can be activated by three kinds of completely different approach：Classical pathway, agglutinin way Footpath and alternative route.Classical pathway is typically the compound triggering being made up of host antibodies combination foreign particle (i.e. antigen), And therefore need to produce specific antibody response exposed to the antigen in advance.Because the activation of classical pathway depends on previous Host Acquired immune response, so classical pathway is a part for acquired immune system.On the contrary, lectin pathway and Both alternative routes are a parts for innate immune system independent of acquired immunity.

The activation of complement system causes the sequential activation of serine protease zymogen.The first step of classical pathway activation is special The opposite sex recognizes molecule C1q and combines the combination of the IgG and IgM compounds of antigen.C1q and C1r and C1s serine protease enzymes Original is combined into referred to as C1 compound.When C1q is combined with immune complex, C1r Arg-Ile sites carry out self albumen water Solution is cut, and is followed by the C1s cuttings of C1r mediations and is activated, it cuts C4 and C2 ability so as to obtain.C4 cuts into two pieces Section, referred to as C4a and C4b, also, similarly, C2 cuts into C2a and C2b.C4b fragments can be with neighbouring hydroxyl or amino shape Into covalent bond, and by generating C3 convertase (C4b2b) with activating C2 C2a fragments progress noncovalent interaction.C3 Invertase (C4b2b) cuts into C3a and C3b subcomponents by proteolysis and activates C3, causes C5 convertase (C4b2a3b) Generation, it by cutting C5 causes that membrane attack complex (C5b combinations C6, C7, C8 that cell membrane causes cell to crack can be destroyed And C9, also referred to as " MAC ") generation.C3 and C4 activated form (C3b and C4b) is covalently deposited on external source target surface, its quilt Complement receptors on a variety of phagocytes is recognized.

Independently, the first step that complement system is activated by lectin pathway is also the combination of specific recognition molecules, its It is followed by the activation of combined serine stretch protein proenzyme.However, the identification molecule in lectin pathway is referred to as including one group Carbohydrate-binding protein (mannan-binding lectin (MBL), H- fiber gelatinized proteins (H-ficolin), the M- viscoses of agglutinin Coagulated protein, L- fiber gelatinized proteins and c-type agglutinin CL-11), rather than immune complex is combined by C1q.Referring to J. Lu etc.,Biochim. Biophys. Acta 1572:387-400, 2002;Holmskov etc.,Annu. Rev. Immunol. 21:547-578 (2003);Teh etc.,Immunology 101:225-232 (2000)).Referring further to J. Luet etc.,Biochim Biophys Acta1572:387-400 (2002);Holmskov etc.,Annu Rev Immunol21:547-578 (2003);Teh etc.,Immunology 101:225-232 (2000); Hansen S. Deng,J. Immunol 185(10):6096-6104 (2010)。

Ikeda et al. is proved first, similar with C1q, MBL with can be with after the coated erythrocyte binding of yeast mannans Made in the way of relying on C4 complement system activation (K. Ikeda etc.,J. Biol. Chem.262:7451-7454,1987). MBL is the member of collectin protein family, is calcium dependent agglutinin, its with being positioned on the pyranose ring equatorial plane 3- hydroxyls and 4- hydroxyls carbohydrate combine.Therefore MBL important part is D-MANNOSE and N- acetyl-GLUCOSAMINE, without Meet the carbohydrate of this space requirement do not have to MBL then detectable compatibility (Weis, W.I. etc.,Nature 360:127- 134,1992).Interaction between MBL and unit price sugar is quite faint, and dissociation constant is generally units person of outstanding talent's mole In the range of.MBL is realized by affinity by being interacted with position multiple monosaccharide residues closely adjacent one another simultaneously Glycan ligand specificity is combined closely (Lee, R.T. etc.,Archiv. Biochem. Biophys.299:129-136, 1992).MBL recognizes usual modified microorganism such as bacterium, yeast, parasite and some viral sugared patterns.On the contrary, MBL is failed to see The sugar of the sugared and last position of other D- galactolipins and sialic acid, i.e. penultimate, they typically modify mammalian plasma Glycoconjugate is combined with " maturation " present on cell surface glycoprotein.Think that this binding specificity promotes " external source " surface Recognize and help to protect against autoactivation.However, MBL is really with high-affinity combination high mannose " precursor " glycan cluster, These clusters, which are located at, to be isolated on the glycoprotein and glycolipid of the N- connections in mammalian cell endoplasmic reticulum and golgiosome (Maynard, Y. etc.,J. Biol. Chem.257:3788-3794,1982).Therefore, damaged cell is combined via MBL Lectin pathway activation potential target.

Fiber gelatinized protein has the Lectin domain different types of with MBL, referred to as fibrinogen spline structure domain. Fiber gelatinized protein is with independent of Ca⁺⁺Mode combine saccharide residue.In people, it has been identified that three kinds of fiber gelatinized protein Type（L- fiber gelatinized proteins, M- fiber gelatinized proteins and H- fiber gelatinized proteins）.L- fiber gelatinized proteins and H- viscoses Both serum fiber gelatinized proteins of coagulated protein are common to have specificity to N- acetyl-GLUCOSAMINE；However, the fiber gelatinized eggs of H- In vain herein in connection with N- acetyl-D-galactosamine.L- fiber gelatinized proteins, H- fiber gelatinized proteins, CL-11 and MBL sugar specificity Difference mean that different agglutinins can be complementary, in spite of overlapping, but different glycoconjugates can be targetted.This is seen Point has obtained the support of nearest report, i.e., in the known agglutinin of lectin pathway, only L- fiber gelatinized proteins and fat phosphorus Teichaic acid is specifically bound, and the lipoteichoicacid is a kind of cell wall sugar conjugate found on all gram-positive bacterias (Lynch, N.J. etc.,J. Immunol. 172:1198-1202,2004).Collectin (i.e. MBL) and fiber gelatinized protein There is no significant similitude on amino acid sequence.However, two histone matter have a similar domain group structure, and with C1q classes Seemingly, oligomeric structure is assembled into, the possibility for so allowing for the combination of multidigit point is maximized.

MBL serum-concentration is alterable height in healthy population, this be in heredity by mbl gene promoter and Polymorphism/mutation of both code areas is controlled.As acute phase protein, MBL expression is dialled further up during inflammation. L- fiber gelatinized proteins concentration present in serum is similar with MBL concentration.Therefore, the fiber gelatinized eggs of the L- of lectin pathway White branch approach may be equally matched with MBL branches in intensity.MBL and fiber gelatinized protein may also be acted as opsonin With, its allow phagocyte target surface that MBL and fiber gelatinized protein are modified (referring to Jack etc.,J Leukoc Biol., 77(3):328-36 (2004);Matsushita and Fujita,Immunobiology, 205(4-5):490-7 (2002);Aoyagi etc.,J Immunol174(1):418-25 (2005)).This opsonification need these albumen with Phagocyte acceptor interaction（Kuhlman, M. etc.,J. Exp. Med.169:1733,1989；Matsushita, M. etc.,J. Biol. Chem.271:2448-54,1996）, the identity of these phagocyte acceptors do not determined also..

People MBL is by the way that its collagen spline structure domain and unique C1r/C1s sample serine proteases form specificity, height is affine Property interaction, serine protease (MASP) related referred to as MBL.It is heretofore described three kinds of MASP.First, Single enzyme " MASP " is identified, and it is characterized in that is used as the enzyme for being responsible for starting complement cascade (cutting C2 and C4) (Matsushita M and Fujita T.,J Exp Med176(6):1497-1502 (1992), Ji, Y.H. etc.,J. Immunol. 150:571-578,1993).It is then determined that MASP activity is actually two kinds of protease MASP-1 and MASP-2 Mixture (Thiel, S. etc.,Nature386:506-510,1997).But, it was demonstrated that MBL-MASP-2 compounds are independent just Be enough to make complement activation (Vorup-Jensen, T. etc.,J. Immunol. 165:2093-2100,2000).In addition, only MASP-2 with high-speed cutting C2 and C4 (Ambrus, G. etc.,J. Immunol.170:1374-1382,2003).Therefore, MASP-2 is responsible for activation C4 and C2 to produce C3 convertase C4b2a protease.This is to be different from C1 in classical pathway to be combined The significant difference of thing, two of which specific serine protease (C1r and C1s), which acts synergistically, result in the work of complement system Change.In addition, isolated the third new protease MASP-3 (Dahl, M.R. etc.,Immunity 15:127-35, 2001).MASP-1 and MASP-3 are the alternative splicing products of same gene.

The shared identical domain group structures of the enzyme component C1r and C1s of MASP and C1 compounds (Sim, R.B. etc.,Biochem. Soc. Trans.28:545,2000).These domains include N-terminal C1r/C1s/ sea urchin VEGF/ bon e formations Albumen (CUB) domain, epidermal growth factor-like domain, the 2nd CUB domains, series connection complement regulatory proteins domain and Serine protease domain.With in C1 protease, MASP-2 activation by serine protease domain near Arg-Ile bond cleavages solution and occur, enzyme is divided into the A chains and B chains of disulfide bond by it, and the latter is by serine protease domain Constitute.Recently, defect (Stengaard-Pedersen, K. etc., New Eng. J. that MASP-2 heredity is determined are described Med. 349:554-560,2003).The mutation of single nucleotide acid causes the Asp-Gly in the domains of CUB 1 to exchange, and causes MASP-2 can not be combined with MBL.

Alternative splice forms of the MBL also with MASP-2, referred to as 19 kDa MBL GAP-associated protein GAPs (MAp19) (Stover, C.M.,J. Immunol.162:3481-90,1999) or small MBL GAP-associated protein GAPs (sMAP) (Takahashi, M. etc.,Int. Immunol. 11:859-863,1999) association, the catalytic activity of the hypoproteinosis MASP-2.MAp19 includes MASP-2 the first two domain, it is followed by the additional sequences of 4 unique amino acids.MASP 1WithMASP 2Gene is located at respectively On No. 3 and No. 1 chromosome of people (Schwaeble, W. etc.,Immunobiology 205:455-466,2002).

Some evidences show there are different MBL-MASP compounds, and the various MASP in serum major part not with MBL it is compound (Thiel, S. etc.,J. Immunol.165:878-887,2000).H- fiber gelatinized proteins and the fiber gelatinized eggs of L- In vain all combined with various MASP, and activate agglutinin complement pathway, as carried out by MBL (Dahl, M.R. etc.,Immunity 15: 127-35,2001;Matsushita, M. etc.,J. Immunol.168:3502-3506,2002).Lectin pathway and warp Allusion quotation approach all forms common C3 convertase (C4b2a), and this two approach merge in this step.

Generally believe that lectin pathway host in inmature host plays an important roll in anti-infectious defence.MBL joins With the strong evidence of host defense come to feature MBL serum levels reduce patient analysis (Kilpatrick, D.C.,Biochim. Biophys. Acta1572:401-413,2002).These patients show recurrent bacterial and fungi The neurological susceptibility of infection.These symptoms from parent generally in life in early days during the apparent window of rapid wear it will be evident that because obtain Antibody titer is reduced, but before complete antibody response storehouse development.This symptom is often due to the number of MBL collagenous portions Caused by individual site mutation, it hampers being properly formed for MBL oligomer.However, because MBL can be used as independent of complement Opsonin work, so it is due to what extent impaired complement activation to have no knowledge about the neurological susceptibility increase to infection It is caused.

With classical pathway and lectin pathway on the contrary, not finding to complete the initiation factor of identification function in alternative route (initiator), it is C1q and agglutinin in other two kinds of approach to complete identification function.At present it is commonly accepted that, The low-level all Transactivations of the spontaneous experience of alternative route（turnover activation）, its can easily in foreign surface or Amplify on other abnormal surfaces (bacterium, yeast, the cell or damaged tissues of virus infection), its independently keep spontaneous activation by The appropriate molecular element of control.There are four kinds of plasma proteins directly to take part in the activation of alternative route：C3, Factor B, the D factors and standby Xie Su.Although a large amount of evidences show in the pathogenesis of non-infectious human diseases, classic complement approach and replacement complement way Both footpath is present, but the evaluation acted on lectin pathway has just just started.Study the evidence provided recently to show, aggegation The activation of plain approach is probably the reason for causing complement activation and related inflammation in ischemia/reperfusion injury.Collard et al. (2000) the endothelial cell combination MBL of the culture by oxidative stress is reported, and shows that C3 is deposited when exposed to human serum (Collard, C.D. etc.,Am. J. Pathol.156:1549-1556,2000).In addition, with the anti-MBL monoclonals of closure Antibody processing human serum inhibits MBL to combine and complement activation.These discoveries are expanded into ischemia-reperfusion rat model On, wherein compared with the rat handled with control antibodies, the rat handled with the blocking antibody for rat MBL is in coronary artery Myocardial damage significantly relatively light (Jordan, J.E. etc., Circulation 104 are shown when inaccessible:1413-1418,2001).Still The molecular mechanism that MBL is combined with blood vessel endothelium after oxidative stress is not known；It has recently been demonstrated that agglutinin way after oxidative stress The activation in footpath is probably to be combined by MBL and mediated with vascular endothelial cell keratin, rather than is combined with glycoconjugate (Collard, C.D. etc.,Am. J. Pathol.159:1045-1054,2001).Other researchs have shown out ischemia/reperfusion The effect of classical pathway and alternative route and lectin pathway in this disease in note damage pathogenesis still has Dispute (Riedermann, N.C. etc.,Am. J. Pathol. 162:363-367,2003).

Recent study, which is shown, needs MASP-1（And it is MASP-3 to be also possible to）By the alternative route activating enzymes D factors from its Zymogen forms change into its enzymatic activity form（Referring to Takahashi M. et al.,J Exp Med 207(1):29-37 (2010)）.The physiological significance of this process is lived by the way that alternative route function is not present in the blood plasma of MASP-1/3 deficient mices Property and emphasized.For alternative route, it is necessary to which the C3b generated by natural C3 proteolysis plays a role.Due to alternative route C3 convertase (C3bBb) contains C3b as required subunit, has carried the problem of on being originated via first C3b of alternative route The problem of it is to have gone out puzzlement, and promoted considerable research work.

C3 belongs to the protein families containing few posttranslational modification for being referred to as thioester bond (with the huge ball eggs of C4 and α -2 Bai Yiqi).Thioester group is made up of glutamine, the sulfydryl of the cysteine of its terminal carbonyl group and three amino acid distances Group forms covalent thioesters connection.The key is unstable, and electrophilic glutamy thioesters can be with nucleophilic moiety such as hydroxyl or ammonia Base radical reaction and thereby with other molecules formation covalent bond.When being isolated in inside complete C3 hydrophobic pocket, thioester bond It is quite stable.However, C3 cuts into C3a and C3b by proteolysis, the thioester bond of high response on C3b is caused to expose Come, and with the nucleophillic attack by the neighbouring part including hydroxyl or amino group, C3b and target covalent bond.It is big except having Measure documentary evidence C3b and outside the covalently bound effect of complement target, also research thinks that C3 thioesters has the pass of triggering alternative route Key is acted on.According to generally accepted " slow-speed theory (tick-over theory) ", alternative route is by liquid-phase conversion enzyme iC3Bb Generation started, iC3Bb is by C3 and hydrolysis thioesters (iC3; C3(H₂O)) and Factor B formation (Lachmann, P.J. Deng Springer Semin.Immunopathol. 7:143-162,1984).C3b sample C3 (H₂O) by natural C3 through protein The slow spontaneous hydrolysis of middle internal thioesters and produce (Pangburn, M.K. etc.,J. Exp. Med.154:856-867, 1981).Pass through C3 (H₂O) the activity of Bb invertases, C3b molecule depositions are on target surface, so as to start alternative route.

The understanding of the initiation factor activated to alternative route is very few.Thinking activation factor, (yeast gathers including yeast cell wall Sugar), many pure polysaccharide, rabbit erythrocyte, some immunoglobulins, virus, fungi, bacterium, animal tumor cell, parasite and Damaged cell.Unique features common to these activation factors are the presence of carbohydrate, but the complexity of carbohydrate structure and various Property make it difficult to determine shared molecule determinant, this is admitted.It is widely accepted that alternative route activation passes through this way Fine equilibrium control between the inhibition modifying ingredients in footpath, such as the H factors, FI, DAF, CR1 and properdin, it is to substitute The only positive regulatory factor of approach.Referring to Schwaeble W.J. and Reid K.B.,Immunol Today 20(1):17- 21 (1999))。

Except obvious unadjusted activation mechanism described above, alternative route can also be agglutinin/classical pathway C3 convertase（C4b2a）The amplifying ring of strength is provided, because the C3b of any generation can participate in forming extra with Factor B Alternative route C3 convertase（C3bBb）.Alternative route C3 convertase is stabilized by combining properdin.Properdin makes replacement way Six to ten times of the Increased Plasma Half-life of footpath C3 convertase.Cause alternative route C5 convertase to alternative route C3 convertase addition C3b Formation.

Think that all three approach (i.e. classical, agglutinin and alternative route) meet at C5 all the time, it is cut shape Into the product with a variety of pro-inflammatory effects.Approach after congregation is referred to as terminal complement approach.C5a is maximally effective allergy poison Element, causes the change of smooth muscle and vascular tone and vasopermeability.It is also neutrophil cell and monocyte two The strong chemotactic factor (CF) and activation factor of person.The cell activation of C5a mediations can discharge a variety of other inflammation by inducing Disease medium carrys out greatly enlarged inflammatory reaction, other inflammatory mediator include cell factor, hydrolase, arachidonic acid metabolite and Active oxygen classification.C5 cracks the formation that result in C5b-9, and it is also referred to as membrane attack complex (MAC).Current strong card According to showing, the MAC depositions of Asia cracking are in addition to plaing a part of to crack pore-forming compound, it is also possible to important work is also played in inflammation With.

Except its important function in immune defense, complement system causes tissue damage in many clinical conditions.Cause This, to developing therapeutically effective complement inhibitor with prevent these ill effects exist it is urgent the need for.

The content of the invention

This general introduction is provided, so as to introduce the selection of concept in simplified form, it is further described in following detailed description of the invention.This is general The key feature for being both not intended to identify claimed theme is stated, the scope for assisting the claimed theme of determination is also not intended to.

In one aspect, the present invention provides the human monoclonal antibodies or its antigen-binding fragment of separation, and it combines people MASP- 2, including（i）Include the weight chain variable district of CDR-H1, CDR-H2 and CDR-H3 sequence；With（ii）Including CDR-L1, CDR-L2 and CDR-L3 light chain variable district, wherein weight chain variable district CDR-H3 sequences include being described as SEQ ID NO:38 or SEQ ID NO: 90 amino acid sequence, and the modification of its conserved sequence, wherein light chain variable district CDR-L3 sequences include being described as SEQ ID NO: 51 or SEQ ID NO:94 amino acid sequence, and the modification of its conserved sequence, and the antibody of wherein described separation suppresses MASP- 2 complement activations relied on.

On the other hand, the present invention provides the human antibody for combining people MASP-2, wherein the antibody includes：I）a）Heavy chain can Become area, it includes i）Including SEQ ID NO：21 amino acid sequence 31-35 heavy chain CDR-H1；And ii）Including SEQ ID NO：21 amino acid sequence 50-65 heavy chain CDR-H2；And iii）Including SEQ ID NO：21 amino acid sequence 95-102's Heavy chain CDR-H3；And b）Light chain variable district, it includes i）Including SEQ ID NO：25 or SEQ ID NO：27 amino acid sequence 24-34 light chain CDR-L1；And ii）Including SEQ ID NO：25 or SEQ ID NO：27 amino acid sequence 50-56 light chain CDR-L2；And iii）Including SEQ ID NO：25 or SEQ ID NO：27 amino acid sequence 89-97 light chain CDR-L3；Or II）With the variable domains in its variant of other side identical, except in the CDR region of the weight chain variable district extremely 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors altogether combined more and 10 ammonia altogether at most combined in the CDR region of the light chain variable district Base acid substitution, wherein the antibody or its variant suppress the complement activation that MASP-2 is relied on.

On the other hand, the present invention provides the human monoclonal antibodies or its antigen binding fragment for the separation for combining people MASP-2 Section, wherein the antibody includes：I）a）Weight chain variable district, it includes i）Including SEQ ID NO：20 amino acid sequence 31-35 Heavy chain CDR-H1；And ii）Including SEQ ID NO：20 amino acid sequence 50-65 heavy chain CDR-H2；And iii）Including SEQ ID NO：18 or SEQ ID NO：20 amino acid sequence 95-102 heavy chain CDR-H3；And b）Light chain variable district, it includes i） Including SEQ ID NO：22 or SEQ ID NO：24 amino acid sequence 24-34 light chain CDR-L1；And ii）Including SEQ ID NO：22 or SEQ ID NO：24 amino acid sequence 50-56 light chain CDR-L2；And iii）Including SEQ ID NO：22 or SEQ ID NO：24 amino acid sequence 89-97 light chain CDR-L3；Or II）With the variable domains other side identical its Variant, except 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors altogether at most combined in the CDR region of the heavy chain and in the light chain variable 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors altogether at most combined in the CDR region in area, wherein the antibody or its variant suppress MASP-2 according to Bad complement activation.

On the other hand, the present invention provides the monoclonal antibody for the separation for combining people MASP-2, or its antigen-binding fragment, Including weight chain variable district, the weight chain variable district includes SEQ ID NO:18、SEQ ID NO:20 or SEQ ID NO:Retouched in 21 Any amino acid sequence stated.

On the other hand, the present invention provides the monoclonal antibody for the separation for combining people MASP-2, or its antigen-binding fragment, Including light chain variable district, the light chain variable district includes SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:25 or SEQ ID NO:Any amino acid sequence described in 27.

On the other hand, the present invention provides the core of the anti-MASP-2 antibody of the coding present invention or the amino acid sequence of its fragment Those described in acid molecule, such as table 2.

On the other hand, the present invention provides the anti-MASP-2 antibody at least including the coding present invention or the amino acid of its fragment The cell of one of the nucleic acid molecules of those described in the nucleic acid molecules of sequence, such as table 2.

On the other hand, the invention provides the method for the MASP-2 antibody of generation separation, it is included in permission code book and sends out Being cultivated under conditions of the expression of nucleic acid of bright anti-MASP-2 antibody at least includes the amino of the anti-MASP-2 antibody of the coding present invention One of nucleic acid molecules of acid sequence cell, and separate the anti-MASP-2 antibody.

On the other hand, the present invention provides human monoclonal antibodies or its antigen-binding fragment of separation, and it is such as to pass through 10nM or lower K that surface plasma body resonant vibration is determined_DFrom people MASP-2 dissociation, and in the coated matrix of mannosan With 10nM or lower IC in 1% serum₅₀Suppress C4 activation.In some embodiments, the antibody or its antigen-binding fragment At least part of epitope recognized by reference antibody of specific recognition, wherein the reference antibody is included such as in SEQ ID NO: Weight chain variable district described in 20 and such as in SEQ ID NO:Light chain variable district described in 24.

On the other hand, the present invention, which is provided, includes the of the invention anti-MASP-2 antibody of human monoclonal and pharmaceutically acceptable The composition of excipient.

On the other hand, the present invention provides the method for suppressing the complement activation that MASP-2 is relied in people experimenter, including To suppress the human monoclonal antibodies that the amount for the complement activation that MASP-2 is relied on applies the present invention enough in the people experimenter.

On the other hand, the present invention provides the product for the unit dose for including the human monoclonal MASP-2 antibody of the present invention, It is suitable for the treatment to people experimenter to apply, wherein the unit dose is 1mg-1000mg scope.

Brief description of the drawings

By reference to following detailed description together with accompanying drawing, foregoing aspect of the invention and many bonus will more It is comprehensible, equally also it is best understood from, wherein：

Figure 1A is the figure of illustration people's MASP-2 genome structures；

Figure 1B is the figure of illustration people's MASP-2 protein structure domain structures；

The result that the ELISA that Fig. 2 illustrations are carried out on polyclonal population as described in Example 2 is determined, it is described polyclonal Colony, which is selected from, is directed to various MASP-2 antigens elutriations（panned）ScFcv phage libraries；

Fig. 3 A and 3B show 45 candidate scFv clones' that functional activity is directed in complement measure as described in example 3 above Test result；

Fig. 4 illustrations carry out comparing three kinds of serum as described in Example 4（People, rat and NHP）The experiment of middle C3c levels As a result；

Fig. 5 A are that as described in Example 4 disclose is belonging respectively to two different groups of VH2 with VH6 gene families most active The heavy chain region of clone（Residue 1-120）Amino acid alignment；

Fig. 5 B are the amino acid alignments that scFv as described in Example 4 clones 17D20,17N16,18L16 and 4D9；

Female clone that IgG4 is converted during the C3b depositions of the human plasma of use 90% of Fig. 6 illustrations as described in example 5 above are determined （mother clone）Prepared product inhibitory activity.

Fig. 7 A illustrations 17N16 mothers clone as described in Example 6 son clone relatively（daughter clone）To people MASP- The result that the ELISA of 2A titration is determined；

Fig. 7 B illustrations 17D20 mothers clone as described in Example 6 son clone relatively surveys to the ELISA that people MASP-2A is titrated Fixed result；

The protein sequence that Fig. 8 is female clone 17N16 and 17N9 clone as described in example 6 above is compared, and shows light chain（From SYE is originated）There is the difference of 17 amino acid residues between being cloned two；

Fig. 9 is clone #35, #59 and #90 of the mutation from compared to 17D20 mother clones as described in example 7 above sequence The protein sequence in CDR-H3 areas is compared；

Figure 10 A are as embodiment 7 resets clone 17D20md21N11 in the CDR3 areas of described 17D20 mother clones with chain and combines The mutant clon #35CDR-H3 clones shown in 17D20md21N11 VL Fig. 9（VH35-VL21N11）Protein sequence ratio It is right；

Figure 10 B are 17D20 mothers clones and the albumen sequence in son clone 17D20md21N11 VL and VH areas as described in example 7 above Row are compared；

MASP-2 monoclonal antibodies anti-to the derived from human mother clone 17N16 of Figure 11 A illustrations as described in example 8 above son Clone isotype variant（MoAb#1-3）The result that the C3b depositions of progress are determined；

MASP-2 monoclonal antibodies anti-to the derived from human mother clone 17D20 of Figure 11 B illustrations as described in example 8 above son Clone isotype variant（MoAb#4-6）The result that the C3b depositions of progress are determined；

Figure 12 A and 12B illustration as described in example 8 above C3b deposit survey female clone for being fixed in 95% serum and MoAb#1-6 test；

The suppression that C4b is deposited in 95% normal human serum as described in example 8 above of Figure 13 illustrations；

The suppression that C3b is deposited in 95% cercopithecus aethiops serum as described in example 8 above of Figure 14 illustrations；

Figure 15 illustrations are cut by the C4 of MoAb#2-6 pre-assembled MBL-MASP-2 compounds as described in example 8 above The suppression of activity；

MoAb#6 compares preferential combinations of the C1s to people MASP-2 to Figure 16 illustrations as described in example 8 above；

The aggegation after the anti-human MoAb#OMS646 of intravenous administration to cercopithecus aethiops of Figure 17 illustrations as described in example 10 above Plain approach is totally constrained；

Figure 18 A are kaplan-Meier survival rates（Kaplan-Meier survival plot ）, in display such as embodiment 11 It is described in control mice and with anti-mouse MASP-2 antibody（mAbM11）Or anti-human MASP-2 antibody（mAbOMS646）The mouse of processing In exposed to 7.0Gy radiate after with the time percent survival；

Figure 18 B are kaplan-Meier survival rates, and display is as described in example 11 above in control mice and with anti-mouse MASP-2 Antibody（mAbM11）Or anti-human MASP-2 antibody（mAbOMS646）With the time after 6.5Gy radiation in the mouse of processing Percent survival；

Figure 18 C are kaplan-Meier survival rates, and display in control mice and uses anti-human MASP-2 as described in example 11 above Antibody（mAbOMS646）Percent survival in the mouse of processing after 8.0Gy radiation with the time；

Figure 19 illustrations are as described in example 12 above in anti-MASP-2 antibody OMS646（Response unit（With reference to）Relative time （In seconds））Upper surface plasma resonance（Biacore）The result of analysis, shows the OMS646 of immobilization with about 1-3x10^-4S^-1 K_offRate and about 1.6-3x10⁶M^-1S^-1K_onRate combines restructuring MASP-2；

The anti-MASP-2 antibody OMS646 of the determination of Figure 20 illustrations as described in example 12 above is to the people MASP-2's of immobilization The result that the ELISA of binding affinity is determined, OMS646 is with about 100pM K for display_DWith reference to immobilization recombined human MASP-2；

Figure 21 A illustrations as described in example 12 above on the coated surface of mannosan presence or absence of anti- MASP-2 antibody（OMS646）In the case of C4 activation levels, it was demonstrated that OMS646 is on the coated surface of mannosan in 1% human blood With about 0.5nM IC in clear₅₀Suppress C4 activation；

Figure 21 B illustrations are as described in example 12 above presence or absence of anti-MASP-2 antibody on the coated surfaces of IgG （OMS646）In the case of C4 activation levels, display OMS646 do not suppress classical pathway dependence complement component C4 activation；

Figure 22 A illustrations are as described in example 12 above under the conditions of lectin pathway specific assay, in existence or non-existence Anti- MASP-2 antibody（OMS646）In the case of MAC deposit level, it was demonstrated that OMS646 is with about 1nM IC₅₀Value suppresses agglutinin The MAC depositions of mediation；

Figure 22 B illustrations under the conditions of classical pathway specific assay, resist in existence or non-existence as described in example 12 above MASP-2 antibody（OMS646）In the case of MAC deposit level, it was demonstrated that OMS646 do not suppress classical pathway mediation MAC sink Product；

Figure 22 C illustrations under the conditions of alternative route specific assay, resist in existence or non-existence as described in example 12 above MASP-2 antibody（OMS646）In the case of MAC deposit level, it was demonstrated that OMS646 do not suppress alternative route mediation MAC sink Product；

Figure 23 A illustrations are being presence or absence of one as described in example 12 above under lectin pathway particular conditions The anti-MASP-2 antibody of row concentration（OMS646）In the case of in 90% human serum C3 deposit level, it was demonstrated that OMS646 is in life C3 depositions are blocked under the conditions of reason；

Figure 23 B illustrations are being presence or absence of one as described in example 12 above under lectin pathway particular conditions The anti-MASP-2 antibody of row concentration（OMS646）In the case of in 90% human serum C4 deposit level, it was demonstrated that OMS646 is in life C4 depositions are blocked under the conditions of reason；

Figure 24 A illustrations under lectin pathway particular conditions, resist in existence or non-existence as described in example 12 above MASP-2 antibody（OMS646）In the case of in 90%Cynomuglus monkey serums C4 deposit level, it was demonstrated that OMS646 exists C4 depositions, the IC with 30-50nM scopes are suppressed in dose response mode in Cynomuglus monkey serums₅₀Value；With

Figure 24 B illustrations under lectin pathway particular conditions, resist in existence or non-existence as described in example 12 above MASP-2 antibody（OMS646）In the case of in 90% cercopithecus aethiops serum C4 deposit level, it was demonstrated that OMS646 Africa it is green C4 depositions, the IC with 15-30nM scopes are suppressed in dose response mode in monkey serum₅₀Value.

The explanation of sequence table

SEQ ID NO:1 people MASP-2 cDNA

SEQ ID NO:2 people's MASP-2 albumen（With leader peptide（leader））

SEQ ID NO:3 people's MASP-2 albumen（Ripe）

SEQ ID NO:4 rat MASP-2 cDNA

SEQ ID NO:5 rat MASP-2 albumen（With leader peptide（leader））

SEQ ID NO:6 rat MASP-2 albumen（Ripe）

Antigen（On people's MASP-2 maturation proteins）

SEQ ID NO:7 people MASP-2 CUBI domains (amino acid/11-121)

SEQ ID NO:8 people MASP-2 CUBI/EGF domains (amino acid/11-166)

SEQ ID NO:9 people MASP-2 CUBI/EGF/CUBII domains (amino acid/11-277)

SEQ ID NO:10 people MASP-2 EGF domains (amino acid/11 22-166)

SEQ ID NO:11 people MASP-2 CCPI/CCPII/SP domains (amino acid 278-671)

SEQ ID NO:12 people MASP-2 CCPI/CCPII domains (amino acid 278-429)

SEQ ID NO:13 people MASP-2 CCPI domains (amino acid 278-347)

SEQ ID NO:14 people MASP-2 CCPII/SP domains (amino acid 348-671)

SEQ ID NO:15 people MASP-2 CCPII domains (amino acid 348-429)

SEQ ID NO:16 people MASP-2 SP domains (amino acid 429-671)

SEQ ID NO:17:The mutant of serine protease inactivation（The amino acid 610-625 of Ser618 with mutation）

Anti- MASP-2 monoclonal antibodies VH chains

SEQ ID NO:18 17D20mc weight chain variable districts (VH) polypeptides

SEQ ID NO:DNA (the no signals of 19 coding 17D20_dc35VH21N11VL (OMS646) weight chain variable districts (VH) Peptide)

SEQ ID NO:20 17D20_dc35VH21N11VL (OMS646) weight chain variable district (VH) polypeptide

SEQ ID NO:21 17N16mc weight chain variable districts (VH) polypeptides

Anti- MASP-2 monoclonal antibodies VL chains

SEQ ID NO:22 17D20mc light chain variable districts (VL) polypeptides

SEQ ID NO:The DNA (no signal peptide) of 23 coding 17D20_dc21N11VL (OMS644) light chain variable districts (VL)

SEQ ID NO:24 17D20_dc21N11VL (OMS644) light chain variable district (VL) polypeptide

SEQ ID NO:25 17D16mc light chain variable districts (VL) polypeptides

SEQ ID NO:The DNA (no signal peptide) of 26 coding 17N16_dc17N9 (OMS641) light chain variable districts (VL)

SEQ ID NO:27 17N16_dc17N9 (OMS641) light chain variable district (VL) polypeptide

Anti- MASP-2 monoclonal antibody heavies CDR

SEQ ID NOS:28-31 CDR-H1

SEQ ID NOS:32-35 CDR-H2

SEQ ID NOS:36-40 CDR-H3

Anti- MASP-2 monoclonal antibodies light chain CDR

SEQ ID NOS:41-45 CDR-L1

SEQ ID NOS:46-50 CDR-L2

SEQ ID NOS:51-54 CDR-L3

MASP-2 antibody sequences

SEQ ID NO:55:ScFv mother's clone's 17D20 full-length polypeptides

SEQ ID NO:56:ScFv mother's clone's 18L16 full-length polypeptides

SEQ ID NO:57:ScFv mother's clone's 4D9 full-length polypeptides

SEQ ID NO:58:ScFv mother's clone's 17L20 full-length polypeptides

SEQ ID NO:59:ScFv mother's clone's 17N16 full-length polypeptides

SEQ ID NO:60:ScFv mother's clone's 3F22 full-length polypeptides

SEQ ID NO:61:ScFv mother's clone's 9P13 full-length polypeptides

SEQ ID NO:62:The DNA of encoding wild type IgG4 heavy chain constant region

SEQ ID NO:63:Wild type IgG4 heavy chain constant region polypeptides

SEQ ID NO:64:The DNA of IgG4 heavy chain constant region of the coding with mutation S228P

SEQ ID NO:65:IgG4 heavy chain constant region polypeptides with mutation S228P

SEQ ID NO:66:ScFv clone's 17N16m_d17N9 full-length polypeptides

SEQ ID NO:67:ScFv clone's 17D20m_d21N11 full-length polypeptides

SEQ ID NO:68:ScFv clone's 17D20m_d3521N11 full-length polypeptides

SEQ ID NO:69:The DNA of encoding wild type IgG2 heavy chain constant region

SEQ ID NO:70:Wild type IgG2 heavy chain constant region polypeptides

SEQ ID NO:71:17N16m_d17N9 light chain genes sequence (has the signal peptide encoded by nucleotides 1-57)

SEQ ID NO:72:17N16m_d17N9 light chain proteins sequence (has signal peptide amino acid/11-19)

SEQ ID NO:73:17N16m_d17N9 IgG2 heavy chain gene sequences (have the signal peptide encoded by nucleotides 1-57)

SEQ ID NO:74:17N16m_d17N9 IgG2 heavy chain proteins sequence (has signal peptide amino acid/11-19)

SEQ ID NO:75:17N16m_d17N9 IgG4 heavy chain gene sequences (have the signal peptide encoded by nucleotides 1-57)

SEQ ID NO:76:17N16m_d17N9 IgG4 heavy chain proteins sequence (has signal peptide amino acid/11-19)

SEQ ID NO:77:The heavy chain gene sequences of 17N16m_d17N9 IgG4 mutation (have the letter encoded by nucleotides 1-57 Number peptide)

SEQ ID NO:78:The heavy chain protein sequence of 17N16m_d17N9 IgG4 mutation (there is signal peptide amino acid/11-19)

SEQ ID NO:79:17D20_3521N11 light chain genes sequence (has the signal peptide encoded by nucleotides 1-57)

SEQ ID NO:80:17D20_3521N11 light chain proteins sequence (has signal peptide amino acid/11-19)

SEQ ID NO:81:17D20_3521N11 IgG2 heavy chain gene sequences (have the signal peptide encoded by nucleotides 1-57)

SEQ ID NO:82:17D20_3521N11 IgG2 heavy chain proteins sequence (has signal peptide amino acid/11-19)

SEQ ID NO:83:17D20_3521N11 IgG4 heavy chain gene sequences (have the signal peptide encoded by nucleotides 1-57)

SEQ ID NO:84:17D20_3521N11 IgG4 heavy chain proteins sequence (has signal peptide amino acid/11-19)

SEQ ID NO:85:The heavy chain gene sequences of 17D20_3521N11 IgG4 mutation (have what is encoded by nucleotides 1-57 Signal peptide)

SEQ ID NO:86:The heavy chain protein sequence of 17D20_3521N11 IgG4 mutation (there is signal peptide amino acid/11-19)

SEQ ID NO:87:The DNA of scFv clone's 17N16m_d17N9 encoding full-length polypeptides（No signal peptide）

SEQ ID NO:88:The DNA of scFv clone's 17D20m_d21N11 encoding full-length polypeptides（No signal peptide）

SEQ ID NO:89:The DNA of scFv clone's 17D20m_d3521N11 encoding full-length polypeptides（No signal peptide）

SEQ ID NO:90:17D20m and d3521N11 shared heavy chain CDR-H3

SEQ ID NO:91:17D20m and d3521N11 shared light chain CDR-L1

SEQ ID NO:92:17N16m and d17N9 shared light chain CDR-L1

SEQ ID NO:93:17D20m, d3521N11,17N16m and d17N9 shared light chain CDR-L2

SEQ ID NO:94:17N16m and d17N9 shared light chain CDR-L3.

Detailed description of the invention

The present invention provide combine people MASP-2 and suppress the complement activation of lectin-mediated and while keeping immune system classical The complete total length human antibody of (C1q is relied on) pathway components.The anti-MASP-2 antibody of people is screened by phage display library It is identified, as described in embodiment 2-9.As described in embodiment 10-12, identify that high-affinity MASP-2 antibody has suppression The ability of the complement activation of lectin-mediated, as external test and in vivoassay are proved.The antibody lightens and heavy chain Fragment is separated with both scFv forms and total length IgG forms.The anti-MASP-2 antibody of people can be used for suppressing lectin-mediated The related cellular damage of complement pathway activation, and the complete of classical (C1q is relied on) pathway components of immune system is kept simultaneously.

I. define

Unless this paper clear stipulaties, otherwise all terms used herein are all with such as the those of ordinary skill institute in field of the present invention The identical meanings of understanding.In order to be specifically used for relevant term of the invention described in this specification and the appended claims, carry For following definition.

Terms used herein " complement activation that MASP-2 is relied on " includes the activation that the MASP-2 of lectin pathway is relied on, its Occur in physiological conditions（That is, there is Ca⁺⁺In the case of）, cause lectin pathway C3 convertase C4b2a formation, and Cause C5 convertase C42a after C3 cleaved products C3b accumulation（C3b）n.

Term " alternative route " as used herein refers to the complement activation for example triggered by zymosan, the zymosan From fungi and yeast cell wall, the lipopolysaccharides (LPS) of Gram-negative outer membrane and rabbit erythrocyte and from a variety of pure many Sugar, rabbit erythrocyte, virus, bacterium, animal tumor cell, parasite and damaged cell, and it is traditionally considered as always It is as caused by the C3b generated from the spontaneous proteolysis of complement factor C_3.

Term " lectin pathway " as used herein refers to via serum and non-serum carbohydrate-binding protein (including mannosan Binding lectin (MBL), CL-11 and fiber gelatinized protein（H- fiber gelatinized proteins, M- fiber gelatinized proteins or L- are fiber gelatinized Albumen）) specific binding and the complement activation that occurs.

It is that term " classical pathway " as used herein refers to be combined and triggered with foreign particle by antibody and need combination Recognize molecule C1q complement activation.

Term " MASP-2 inhibiting antibodies " as used herein refers to any anti-MASP-2 antibody or its MASP-2 binding fragment, It combines or the complement activation of MASP-2 dependences is directly interacted and effectively suppressed with MASP-2.For the inventive method The complement activation that MASP-2 inhibiting antibodies can reduce MASP-2 dependences is more than 20%, is greater than 30%, or more than 40%, or greatly In 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90%, or more than 95%.

Term " MASP-2 blocking antibodies " as used herein refers to MASP-2 inhibiting antibodies, and it reduces the benefit that MASP-2 is relied on Body activation is more than 90%, is greater than 95% or more than 98%（Cause only 10% MASP-2 complement activations, such as only 9%, or only 8%, or only 7%, or only 6%, such as only 5% or less, or only 4%, or only 4%, or only 3%, or only 2% or only 1%）.

Term " antibody " and " immunoglobulin " used interchangeably herein.These terms are that those skilled in the art are abundant Understand, and refer to the albumen being made up of the polypeptide of one or more molecule of the antigen binding.A kind of antibody formation constitutes antibody Basic structural unit.This form is the tetramer and is made up of two pairs of identical antibody chains that each pair has an one light and weight Chain.Per centering, light and weight chain variable district is responsible for combining antigen together, and constant region is responsible for antibody mediated effect thing function.

Term " antibody " as used herein include derived from produce antibody any mammal (such as mouse, rat, rabbit and Primate including people), or derived from hybridoma, phage selection, recombination expression or transgenic animals（Or it is other The method for producing antibody or antibody fragment）Antibody and its antibody fragment, its specific binding with MASP-2 polypeptides or part thereof. Term " antibody " is in antibody sources or the mode of its manufacture（For example, passing through hybridoma, phage selection, recombination expression, transgenosis Animal, peptide symthesis etc.）Aspect is not intended to restricted.Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies；It is more special Heterogenetic antibody（For example, bispecific antibody）；Humanized antibody；Mouse antibody；Chi-meric mice people, mouse primate, primate move Thing human monoclonal antibodies；And anti-idiotype, and can be any entire molecule or its fragment.Term as used herein " antibody " includes not only intact polyclonal or monoclonal antibody, also has its fragment（Such as dAb, Fab, Fab', F (ab')₂、Fv）, It is single-stranded（scFv）, it synthesizes variant, the variant naturally occurred, including the antibody portion with required specific antigen-binding fragment Point fusion protein, humanized antibody, chimeric antibody and it is any other include have needed for specific antigen binding site or Fragment（Epitope recognition site）Immunoglobulin molecules modification configuration.

Term " antigen-binding fragment " as used herein refers to the immunoglobulin weight that people MASP-2 is combined comprising at least one And/or the CDR of light chain polypeptide fragment.Thus, the antigen-binding fragment of antibody described herein can include being self-bonded 1,2,3,4,5 or all 6 CDR of the VH and VL sequences described herein of MASP-2 antibody.MASP-2 described herein is special The antigen-binding fragment of property antibody can combine MASP-2.In certain embodiments, antigen-binding fragment or including antigen knot Close the suppression of the complement activation of the antibody-mediated MASP-2 dependences of fragment.

Term " anti-MASP-2 monoclonal antibodies " as used herein refers to homologous antibody group, wherein the monoclonal antibody by Selection combines the amino acid composition being related in the epitope on MASP-2.Anti- MASP-2 monoclonal antibodies are high to MASP-2 target antigens Degree is specific.Term " monoclonal antibody " not only includes complete monoclonal antibody and full length monoclonal antibodies, also has its piece Section（Such as Fab, Fab', F (ab')₂, Fv）, it is single-stranded（scFv）, its variant includes the fusion protein of antigen-binding portion thereof, Humanized monoclonal antibodies, chimeric mAb and any other ability including the specificity with needed for and combination epitope Antigen-binding fragment（Epitope recognition site）Immunoglobulin molecules modification configuration.

As used herein, the qualifier " monoclonal " indicates the spy for the antibody that basically homologous antibody population is obtained Levy, and be not intended in antibody sources or its manufacture（For example pass through hybridoma, phage selection, recombination expression, transgenosis Animal etc.）Aspect is restricted.The term includes the whole immunoglobulin that superincumbent " antibody " defines lower description, and fragment Deng.Monoclonal antibody can use any technology to obtain, and the technology provides a mean for the continuous cell line in culture medium, example Such as Kohler, G., etc.,Nature 256:The hybridomas of 495,1975 descriptions produce antibody molecules, or they can be with Manufactured by recombinant DNA method（See, e.g. the U.S. Patent number 4,816,567 for authorizing Cabilly）.Monoclonal antibody can be with Using Clackson, T., etc.,Nature 352:624-628,1991, and Marks, J.D., etc.,J. Mol. Biol. 222:Technology described in 581-597,1991 is separated from phage antibody library.These antibody can be any exempt from Epidemic disease globulin type, including IgG, IgM, IgE, IgA, IgD and its any subclass.

The immunoglobulin polypeptides of identification include κ and lambda light chain, and α, γ（IgG1、IgG2、IgG3、IgG4）, δ, ε and μ weight Equivalent in chain or other species.Full-length immunoglobulin " light chain "（About 25kDa or about 214 amino acid）It is included in NH₂ The variable region of the amino acid of end about 110 and κ the or λ constant regions in COOH ends.Full-length immunoglobulin " heavy chain "（About 50kDa or about 446 amino acid）Variable region is similarly included（About 116 amino acid）One of with above-mentioned heavy chain constant region, for example γ（About 330 amino acid）.

Four basic chain antibody units are heterologous tetramer glycoprotein, light by two identicals（L）Chain and two identicals Weight（H）Chain is constituted.IgM antibody is referred to as J chains by 5 substantially heterologous tetramer units and extra polypeptide and constituted, and so as to wrap Containing 10 antigen binding sites.The IgA antibody of secretion, which can polymerize, to be formed multivalence and gathers, including 2-5 basic 4 chain elements together with J chains.Each L chains connect H chains by a covalent disulfide bonds, and two H chains by one or more disulfide bonds each other, take Certainly in H chain isotypes.Every H and L chain also has the intrachain disulfide bond of regular intervals.VH and VL pairing forms single anti-together Former binding site.

Every H chains have variable domains in N-terminal（VH）, it is then three constant domains for every α and γ chain （CH）, it is then four CH domains for μ and ε isotypes（CH）.

Every L chains have variable domains in N-terminal（VL）, it is then constant domain in its other end（CL）.VL with VH aligns, and first constant domain of CL and heavy chain（CH1）Alignment.Based on its constant domain（CL）Amino acid sequence Row, the L chains from any invertebrate species can be assigned to one of two completely different types, referred to as Kappa（κ）And drawing Mu Da（λ）.

Depending on their heavy chains（CH）Constant domain amino acid sequence, immunoglobulin can be assigned to inhomogeneity Type or isotype.There is the immunoglobulin of 5 types：IgA, IgD, IgE, IgG and IgM, which have, is referred to as Alpha (α), moral Your tower (δ), Ai Pusilong (ε), gamma (γ) and wrong (μ) heavy chain.Based on difference and function small in CH sequences, γ and α Type is further separated into subclass, and such as people expresses following subclass：IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

For the structure and property of different types of antibody, see, e.g.Basic and Clinical Immunology, 8th Edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow Editor；Appleton and Lange, Norwalk, Conn., page 1994,71 and the 6th chapter.

Term " variable " refers to the section in some V structure domains the fact changing very big between antibody in terms of sequence.V structure domain Mediate antigen combines and limits specificity of the specific antibodies to its specific antigen.But, 110 in variable domains of changeability It is not evenly distributed in amino acid span.More precisely, V areas by the shorter area of extreme changeability by being referred to as " hypervariable region " （9-12 amino acid of each length）Relatively constant section of the 15-30 amino acid separated is referred to as framework region（FR）Composition.Naturally The variable domains of weight and light chain each include four FR, largely take beta sheet conformation, are connected by three hypervariable regions, its Ring connection is formed, and forms part n- foldable structures in some cases.The hypervariable region of every chain is by FR close to guarantor Hold together, and facilitate with the hypervariable region from other chains the formation of the antigen binding site of antibody（Referring to Kabat, etc.,Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md (1991)）.Constant domain is not direct The combination of antibody and antigen is participated in, but shows various effector functions, cell toxicant of such as antibody in the cell of antibody-dependant Property（ADCC）In participation.

As used herein, term " hypervariable region " refers to the antibody amino acid residue of responsible antigen binding.Hypervariable region is generally included Amino acid residue from " complementary determining region " or " CDR "（That is, when being numbered according to Kabat numbering systems, from light chain Residue about 24-34 in variable domains（L1）、50-56（L2）And 89-97（L3）, and about 31- in heavy-chain variable domains 35（H1）、50-65（H2）And 95-102（H3）, the numbering system such as in Kabat, etc.,Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes Of Health, Bethesda, Md (1991)) described in）；And/or those come from the residue of " hypervariable loop "（That is, basis is worked as When Chothia numbering systems are numbered, residue 24-34 in light variable domains（L1）、50-56（L2）And 89-97（L3）, With 26-32 in heavy-chain variable domains（H1）、52-56（H2）And 95-101（H3）, the numbering system such as in Chothia and Lesk, J. Mol. Biol. 196:Described in 901-917 (1987)）；And/or those come from " hypervariable loop "/CDR residue （For example, when being numbered according to IMGT numbering systems, residue 27-38 in VL（L1）、56-65（L2）And 105-120（L3）, With 27-38 in VH（H1）、56-65（H2）And 105-120（H3）, the numbering system such as in Lefranc, J.P., etc.,Nucleic Acids Res 27:209-212;Ruiz, M., etc.,Nucleic Acids Res 28:219-221 (2000) described in）.

Term " antibody fragment " as used herein refers to the part for being derived from or being related to the anti-MASP-2 antibody of total length, generally includes Its antigen binding or variable region.The illustrative example of antibody fragment includes Fab, Fab', F (ab) 2, F (ab') 2 and Fv fragments, ScFv fragments, double antibody（diabodies）, linear antibodies, single-chain antibody molecules, antibody fragment formation bispecific antibody and Multi-specificity antibody.

When using bispecific antibody, these can be conventional bispecific antibody, and it can be manufactured in various ways （Holliger, P. and Winter G.Current Opinion Biotechnol. 4, 446-449 (1993)）, for example Chemical preparation or from hybridoma, or can be any bispecific antibody fragment mentioned above.

" scFv " or " scFv " antibody fragment includes VH the and VL domains of antibody, wherein these structures as used herein Domain exists with wall scroll polypeptide chain.Generally, Fv polypeptides further comprise the peptide linker between VH and VL domains, and it causes scFv Desired structure, which can be formed, is used for antibody binding.Exist referring to PluckthunThe Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, In New York, pp. 269-315 (1994)." Fv " is the minimum antibody piece for including complete antigen recognizing and binding site Section.This fragment is made up of a weight and a light chain variable domain with the dimer being closely not covalently linked.From this two The folding of individual domain sends six hypervariable loops（Respectively from three rings of H and L chains）, it contributes the amino for antigen binding Sour residue, and antibody is assigned with antigen-binding specificity.But, although the affinity than whole binding site is lower, but i.e. Make single variable domains（Or only include 3 CDR specific to antigen Fv half）Also have and recognize and combine antigen Ability.

Term " specific binding " as used herein refers to antibody and preferentially combines the homogeneous mixture for being present in different analytes In specific analyte ability.In certain embodiments, specific binding, which interacts, will distinguish desired by sample With undesirable analyte, in some embodiments, more than 10-100 times or more（For example more than 1000 or 10000 times）. In certain embodiments, when they are specifically bound in capturing agent/analyte complex, between capturing agent and analyte Affinity be characterized as less than about 100nM, or less than about 50nM, or less than about 25nM, or less than about 10nM, or less than about 5nM, Or less than about 1nM K_D（Dissociation constant）.

The anti-MASP-2 antibody of term " variant " as used herein refer to by adding, lack in parent antibody sequence and/or One or more amino acid residues are replaced to make it different from " parent " or reference antibody amino acid sequence in terms of amino acid sequence Molecule.In one embodiment, the anti-MASP-2 antibody of variant, which refers to, includes and parent's variable domains identical variable region Molecule, except combined in the CDR region of weight chain variable district altogether 1,2,3,4,5,6,7,8,9 or 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and/ Or at most light chain variable district the CDR region have combination altogether 1,2,3,4,5,6,7,8,9 or 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors. In some embodiments, the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is conserved sequence modification.

Term " parental antibody " as used herein refers to the amino acid sequences encoded antibody by being prepared for variant.It is preferred that Ground, the parental antibody have people's framework region and, if it does, with one or more human antibody constant regions.For example, the parent This antibody can be humanization or human antibody.

Term " antibody of separation " as used herein refers to from the Components identification of its natural surroundings and resisting for separating and/or reclaim Body.The composition of the pollution of its natural surroundings is the material by the diagnosis of interference antibody or therapeutical uses, and can include enzyme, swash Plain and other Proteinaceous or non-protein solute.In preferred embodiments, the antibody will be purified（1）To higher than 95 weights % antibody is measured, is such as determined by Lowry methods, and most preferably greater than 99 weight %；（2）To being enough to be sequenced by using rotary-cup type Instrument obtains the degree of at least 15 N-terminal residues or internal amino acid sequence；Or（3）To use Coomassie brilliant blue or preferred silver staining Pass through SDS-PAGE homogeney under reduction or non reducing conditions.The antibody of separation includes antibody in situ in recombinant cell, Because there will be at least one composition of the natural surroundings of antibody.But generally, the antibody of separation will be purified by least one It is prepared by step.

Term " epitope " as used herein refers to the antigen part of monoclonal antibody specificity combination thereon.Epitopic determinants lead to Often it is made up of the chemically reactive surface group of molecule, such as amino acid or sugared side chain, and generally there is specific three-dimensional structure Feature, and specific charge characteristic.More specifically, term " MASP-2 epitopes " as used herein refers to such as by any this area Well known method（For example pass through immunoassays）Determine, the part of the corresponding polypeptide of antibody mediated immunity specific binding.Antigenicity Epitope need not immunogenicity.These epitopes can be actually linear, or can be discontinuous epitope.Therefore, such as Terms used herein " comformational epitope " refers to the spatial relationship rather than a series of continuous amino acid between the amino acid by antigen The discontinuous epitope formed.

Term " mannan-binding lectin " as used herein（“MBL”）It is equivalent to maltose-binding protein （“MBP”）.

As used herein " membrane attack complex "（“MAC”）Refer to the 5 kinds of terminal complement compositions (C5-C9) for inserting and destroying film Compound.Also known as C5b-9.

As used herein " subject " include all mammals, including but not limited to people, non-human primate, Dog, cat, horse, sheep, goat, ox, rabbit, pig and rodent.

The abbreviation of amino acid residue used herein is as follows：Alanine (Ala；A), asparagine (Asn；N), aspartic acid (Asp；D), arginine (Arg；R), cysteine (Cys;C), glutamic acid (Glu；E), glutamine (Gln；Q), glycine (Gly；G), histidine (His；H), isoleucine (Ile；I), leucine (Leu；L), lysine (Lys；K), methionine (Met；M), phenylalanine (Phe；F), proline (Pro；P), serine (Ser；S), threonine (Thr；T), tryptophan (Trp； W), tyrosine (Tyr；) and alanine (Val Y；V).

From most wide meaning, naturally occurring amino acid can be grouped according to the chemical characteristic of each amino acid side chain. " hydrophobic " amino acid refers to Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro." hydrophilic " amino acid refer to Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His.This group of amino acid can be further broken down as follows.It is " not charged Lotus it is hydrophilic " amino acid refers to Ser, Thr, Asn or Gln." acidity " amino acid refers to Glu or Asp." alkalescence " amino acid is Refer to Lys, Arg or His.

Terms used herein " conservative 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor " is illustrated by the substitution between amino acid in following every group： (1) glycine, alanine, valine, leucine and isoleucine；(2) phenylalanine, tyrosine and tryptophan；(3) serine And threonine；(4) aspartic acid and glutamic acid；(5) glutamine and asparagine；(6) lysine, arginine and histidine.

Term " nucleotides of separation " as used herein refers to the nucleic acid molecules for the genomic DNA for not being integrated into organism（Example Such as polynucleotides）.For example, the DNA molecular for the growth factor that coding is separated with the genomic DNA of cell is the DNA molecular separated. Another example of the nucleotides of separation refers to the nucleic acid molecules for not being integrated into the chemical synthesis of the genome of organism.It is isolated from The nucleic acid molecules of particular species are less than the complete DNA molecule of the chromosome from the species.

As used herein " nucleic acid molecules construction " be or single or double chain nucleic acid molecules, its by people intervene modification with Include natural non-existent combination or the nucleic acid segment arranged side by side.

" expression vector " is the nucleic acid molecules for the gene that coding is expressed in host cell as used herein.Typically, table Include transcripting promoter, gene and transcription terminator up to carrier.Gene expression is usually placed under the control of promoter, and the base Because referred to as " being operably connected to " promoter.Similarly, if regulating element adjusts the activity of core promoter, regulation member Part and core promoter are operably connected.

The term " approximate " as used herein for being related to numeral or " about " are generally used with including falling into the digital both direction （It is more than or less than）The numeral of 5% scope, it is unless otherwise indicated or another on evidence from context（Except when the numeral will exceed can Can value 100% when）.It is unless otherwise indicated or another on evidence from context in the range of end points is included in when scope of a declaration.

As used herein, singulative " one ", " one " and "the" include plural number aspect, unless context is clearly in addition Indicate.Thus, for example referring to that " cell " includes individual cells, and two or more cells, refer to that " reagent " is wrapped Include a reagent and two or more reagents；Refer to that " antibody " includes these multiple antibody and refers to " a framework Area " includes referring to one or more framework regions and its equivalent well known by persons skilled in the art, etc..

Each embodiment will make necessary modification in detail applied to each other embodiments in this specification, remove It is non-to be otherwise noted.

For recombinant DNA, oligonucleotide synthesis and tissue cultures and conversion（For example, electricity conversion, lipofection）It can make Use standard technique.Zymetology is reacted and purification technique can be carried out or such as entering that this area is routinely realized according to the explanation of manufacturer OK, or it is as described herein carry out.These and related technology and method can generally enter according to conventional method well known in the art OK, and as this specification quote and discuss in the whole text it is various and more specifically quote described in progress.See, e.g. Sambrook etc., 2001,MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY)；Or other related Current Protocol publication and other similar bibliography.Remove Non- offer is specifically defined, molecular biology described herein, analytical chemistry, synthetic organic chemistry and medical science and pharmaceutical chemical reality Room methods and techniques and used related nomenclature are tested, are that those skilled in the art know and conventional use of.For restructuring Technology, molecular biology, microbiology, chemical synthesis, chemical analysis, medicine preparation, preparation and delivering and patient's treatment can be with Use standard technique.

Any method, kit, reagent or composition on the present invention, it is contemplated that any implementation discussed in this description Scheme can be carried out, and vice versa.Further, composition of the invention can be used for the side for realizing the present invention Method.

II. summarize

Agglutinin（MBL, M- fiber gelatinized protein, H- fiber gelatinized proteins, L- fiber gelatinized proteins and CL-11）It is specific knowledge Other molecule, it triggers congenital complement system and the system includes agglutinin and triggers approach and associated ends approach amplifying ring, It amplifies the activation for the terminal complement effector molecule that agglutinin triggers.C1q is specific recognition molecules, and it triggers acquired mend System is united and the system includes classical initiation approach and associated ends approach amplifying ring, and it amplifies the terminal complement that C1q triggers The activation of effector molecule.We by both main complement activation systems be referred to as Lectin-dependent complement system and The complement system that C1q is relied on.

Except its important function in immune defense, complement system causes tissue damage in many clinical conditions.Cause This, to developing therapeutically effective complement inhibitor with prevent these ill effects exist it is urgent the need for.

Such as U.S. Patent number 7,919,094, Co-pending U.S. Patent Application series number 12/905,972（It is disclosed as US 2011/0091450）With Co-pending U.S. Patent Application series number 13/083,441（It is disclosed as US2011/0311549） （Each of which belongs to Omeros Corporation, present assignee, and each of which and is incorporated herein by reference）In It is described, by using MASP-2 -/- mouse model determine that the MASP-2 approach of lectin-mediated can be suppressed while keeping classical Approach is complete., then can be with based on the MASP-2 paths that can suppress lectin-mediated while keeping the complete accreditation of classical pathway Recognize, specificity only suppresses to cause the immune defense ability of the complement activation of particular pathologies and not fully closed complement, and it will It is very desirable.The morbid state that for example complement activation is mainly mediated by the complement system of Lectin-dependent wherein In, it will be favourable that specificity, which only suppresses this system,.This will keep the integrality of the complement activation system of C1q dependences to exempt to handle Host defense against infection is processed and helped to epidemic disease compound.

In the research and development for the medicine for suppressing the complement system of Lectin-dependent in specificity, preferably as the egg of target Bai Chengfen is MASP-2.In all known protein ingredient (MBL, H- fiber gelatinized protein, the M- of the complement system of Lectin-dependent Fiber gelatinized protein, L- fiber gelatinized proteins, MASP-2, C2-C9, Factor B, the D factors and properdin) in, only MASP-2 is The complement system institute of Lectin-dependent is exclusive and is necessary to this system works.(MBL, H- are fiber gelatinized for agglutinin Albumen, M- fiber gelatinized proteins and L- fiber gelatinized proteins and CL-11) be also Lectin-dependent complement system in it is exclusive into Point.However, any forfeiture of these agglutinin compositions might not suppression system activation, this is due to agglutinin redundancy institute Cause.In order to ensure a suppression of the complement activation system of Lectin-dependent, it will be necessary to suppress all 5 kinds of agglutinins.Further, since It is known that MBL and fiber gelatinized protein have the opsonic activity independent of complement, therefore suppress lectins function will cause funeral Lose this beneficial anti-infectious host defense mechanism.On the contrary, if MASP-2 is suppression target, it is this independent of benefit The agglutinin opsonic activity of body can keep complete.MASP-2 is used as the therapeutic target for the complement activation system for suppressing Lectin-dependent Added advantage is that MASP-2 plasma concentration is in all complement proteins minimum (about 500 ng/ml)；Therefore, to have obtained It is complete to suppress that relatively low intensity of high-affinity MASP-2 inhibitor is only needed to, as proved in embodiment hereof.

As described herein according to foregoing, the present invention provides with high-affinity combination people MASP-2 and can suppress aggegation The anti-MASP-2 antibody of the full people of monoclonal of the complement pathway activation of element mediation.

III. MASP-2 inhibiting antibodies

On the one hand, the present invention provides the anti-MASP-2 antibody of the full people of monoclonal, or its antigen-binding fragment, and it specifically binds people MASP-2 and the complement activation for suppressing or blocking MASP-2 to rely on.MASP-2 inhibiting antibodies can be by suppressing or blocking MASP-2 biological function, the complement activation system for effectively suppressing or effectively blocking MASP-2 to rely on.For example, inhibition Antibody can effectively suppress or block MASP-2 protein-protein interactions, interference MASP-2 dimerizations or assembling, block Ca2+ is combined, or the serine protease activated sites of interference MASP-2.

MASP-2 epitopes

The present invention provides specific binding people MASP-2 human antibody.MASP-2 polypeptide displays are similar to MASP-1, MASP-3 With C1r and C1s（The protease of C1 complement systems）Molecular structure.SEQ ID NO:CDNA molecule encodings MASP- described in 1 2 representational examples（By SEQ ID NO:Amino acid sequence composition described in 2）And provide with targeting sequencing（1-15 ammonia Base acid）People's MASP-2 polypeptides, the targeting sequencing after secretion be cut produce mature form people MASP-2（SEQ ID NO:3）.As shown in Figure 1A, peopleMASP-2Gene includes 12 extrons.People MASP-2cDNA by extron B, C, D, F, G, H, I, J, K and L are encoded.SEQ ID NO:CDNA molecule encoding rats MASP-2 described in 4（By SEQ ID NO:Described in 5 Amino acid sequence is constituted）And the rat MASP-2 polypeptides with targeting sequencing are provided, the targeting sequencing is cut after secretion Produce the rat MASP-2 of mature form（SEQ ID NO:6）.

It would be recognized by those skilled in the art that SEQ ID NO:1 and SEQ ID NO:Sequence disclosed in 4 represents people respectively With rat MASP-2 single allele, and expected occur allelic variation and alternative splicing.SEQ ID NO:1 He SEQ ID NO:The allele variant of the nucleotide sequence shown in 4, including those include silent mutation and those are therein The variant that mutations result in amino acid sequence changes, within the scope of the present invention.The allele variant of MASP-2 sequences can be with Cloned according to standard method by probe cDNA or genomic library from Different Individual.

People's MASP-2 albumen（SEQ ID NO:3）Domain be shown in Figure 1B and table 1 below, and including N-terminal C1r/C1s/ sea urchin VEGF/ BMPs (CUBI) domain, epidermal growth factor-like domain, the 2nd CUB domains （CUBII）, and complement regulatory proteins the domain C CP1 and CCP2 connected, and serine protease domain.MASP-2 bases The alternative splicing of cause produces MAp19.MAp19 is the N-terminal CUB1-EGF areas comprising MASP-2 and four additional residues （EQSL）Non- zymoprotein.

Show that multiple albumen are combined or interacted with MASP-2 by protein-protein interaction.For example it is known MASP-2 is combined and is formed Ca with agglutinant protein MBL, H fiber gelatinized protein and L fiber gelatinized proteins²⁺The compound of dependence. Show that each MASP-2/ agglutinins compound passes through the MASP-2 PROTEIN Cs 4 relied on and C2 cutter activation complement（Ikeda, Etc., K.,J. Biol. Chem. 262:7451-7454, 1987;Matsushita, M., etc.,J. Exp. Med.176:1497-2284, 2000;Matsushita, M., etc.,J. Immunol. 168:3502-3506, 2002）.Research has shown that MASP-2 CUB1-EGF domains are required for MASP-2 and MBL connection （Thielens, N.M., et al.,J. Immunol. 166:5068, 2001）.Have also shown CUB1EGFCUBII knots Structure domain mediates MASP-2 dimerization, and it is formed required for active MBL compounds（Wallis, R., et al.,J. Biol. Chem. 275:30962-30969, 2000）.It therefore, it can identification and the known complement relied on for MASP-2 The MASP-2 inhibiting antibodies that the important MASP-2 target regions of activation are combined or interacted.

Table 1：MASP-2 polypeptide domains

In one embodiment, anti-MASP-2 inhibiting antibodies combination total length people's MASP-2 albumen of the invention（SEQ ID NO:3）Part such as MASP-2 CUBI, EGF, CUBII, CCPI, CCPII or SP.In some embodiments, this hair The epitope of bright anti-MASP-2 inhibiting antibodies combination CCP1 domains（SEQ ID NO:13（SEQ ID NO:3 amino acid 278-347））.For example, as described in example 9 above, anti-MASP-2 antibody（Such as OMS646）Be accredited as only in conjunction with comprising The MASP-2 fragments of CCP1 domains and the complement activation for suppressing MASP-2 dependences.

The binding affinity of MASP-2 inhibiting antibodies

Anti- MASP-2 inhibiting antibodies specific binding people MASP-2（Such as SEQ ID NO:Described by 3, by SEQ ID NO:1 compiles Code）, with affinity of high at least 10 times than other antigens in complement system.In some embodiments, MASP-2 inhibitions Antibody specificity combination people MASP-2, with affinity of high at least 100 times than other antigens in complement system.

In some embodiments, MASP-2 inhibiting antibodies specific binding people MASP-2, with K_D（Dissociation constant） Less than about 100nM, or less than about 50nM, or less than about 25nM, or less than about 10nM, or less than about 5nM, or less than or equal to about 1nM, or less than or equal to 0.1nM.As described in embodiment hereof 3-5, the binding affinities of MASP-2 inhibiting antibodies can be with Using suitable combination mensuration known in the art, such as ELISA determines to determine.

The potency of MASP-2 inhibiting antibodies

In one embodiment, when being measured in 1% serum, MASP-2 inhibiting antibodies be sufficient to effectively suppress MASP-2 according to Bad complement activation, with IC₅₀≤ 30 nM, are preferably less than or about 20nM, or less than about 10nM or less than about 5nM, or less than or Equal to about 3nM, or less than or equal to about 1nM.

In one embodiment, when being measured in 90% serum, MASP-2 inhibiting antibodies are sufficient to effectively suppress The complement activation that MASP-2 is relied on, with IC₅₀≤ 30 nM, are preferably less than or about 20nM, or less than about 10nM or less than about 5nM, Or less than or equal to about 3nM, or less than or equal to about 1nM.

The suppression for the complement activation that MASP-2 is relied on is characterized as what is occurred as the result for applying MASP-2 inhibiting antibodies, One of at least following change in complement system composition：MASP-2 rely on complement activation system product C4a, C3a, C5a and/or C5b9（MAC）（For example, being measured as described in the embodiment 2 of U.S. Patent number 7,919,094）, and their catabolism drop Solve product（For example, C3desArg）Generation or generation suppression, C4 cutting and C4b deposition（For example, as described in example 5 above Measurement）, and its follow-up catabolism catabolite（For example, C4bc or C4d）Reduction, or C3 cutting and C3b deposition（For example, Measure as described in example 5 above）, or its follow-up catabolism catabolite（For example, C3bc or C3d etc.）Reduction.

In some embodiments, MASP-2 inhibiting antibodies of the invention can suppress C3 in whole serum and deposit to pair According to C3 deposit be less than 80%, such as less than 70%, such as less than 60%, such as less than 50%, such as less than 40%, for example less than 30%, such as less than 20%, such as less than 15%, such as less than 10%.

In some embodiments, MASP-2 inhibiting antibodies of the invention can suppress C4 in whole serum and deposit to pair According to C4 deposit be less than 80%, such as less than 70%, such as less than 60%, such as less than 50%, such as less than 40%, for example less than 30%, such as less than 20%, such as less than 15%, such as less than 10%.

In some embodiments, anti-MASP-2 inhibiting antibodies selective depression MASP-2 complement activations（That is, with than At least 100 times or higher of C1r or C1s affinity combination MASP-2）, and the complement activation systemic-function for keeping C1q to rely on is complete It is whole（Retain at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 100% classical pathway activity）.

In some embodiments, the anti-MASP-2 inhibiting antibodies of theme have following features：（a）To people MASP-2 height Affinity（Such as 10nM or lower K_D, preferably 1nM or lower K_D）, and（b）With 30nM or lower in 90% human serum IC₅₀, preferably 10nM or lower IC₅₀Suppress the complement activation that MASP-2 is relied on.

As described in embodiment 2-12, the complement of lectin-mediated has been identified with high-affinity combination MASP-2 and suppressed Activate and keep simultaneously the complete total length human antibody of classics (C1q is relied on) pathway components of immune system.The antibody It can lighten and be sequenced, separate and analyze with both scFv forms and total length IgG forms with heavy chain fragment.Fig. 5 A are seven scFv The amino acid alignment of anti-MASP-2 clones, the clone identification suppresses to have binding affinity to MASP-2 and having The active ability that MASP-2 is relied on.Fig. 5 B are four scFv mother clones 17D20,17N16,18L16 and 4D9 amino acid sequence Compare, display frame area and CDR region.As described in embodiment 6 and 7, scFv mother clone 17D20 and 17N16 receive affinity into It is ripe, cause compared to generation of female clone with more high-affinity and the increased sub- clone of potency.ScFv grams shown in Fig. 5 A and 5B The weight chain variable district of grand and produced son clone（VH）（Amino acid/11-120）And light chain variable district（VL）（Amino acid/11 48- 250）Amino acid sequence be shown in table 2 below.

By comparing the weight and light-chain amino acid sequence of anti-MASP-2 inhibiting antibodies discussed above, and determine that those resist Which amino acid occurs for which position of body, discloses the position of substitution of the anti-MASP-2 inhibiting antibodies of people, and can replace Enter the selection of the amino acid of those positions.In an exemplary embodiment, the weight and light chain amino than right Fig. 5 A and 5B Acid sequence, and the amino acid identity of each position of exemplary antibodies is determined.As explained in Fig. 5 A and 5B（Explaination is shown The amino acid that the weight of example property MASP-2 inhibiting antibodies and each position of light chain are present）, easily identifying several can take The position in generation, and the amino acid residue into these positions can be substituted.In another exemplary embodiment, than right Female and son clone light-chain amino acid sequence, and the amino acid identity of each position of exemplary antibodies is determined, can with determination With substituted position, and the amino acid residue into these positions can be substituted.

Table 2：The sequence of representative anti-MASP-2 antibody

In certain embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people has and any described in table 2 Heavy-chain variable domains sequence is substantially the same（For example, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least About 90%, at least about 95%, or at least about 96% is identical, or at least about 97% identical, or at least about 98% is identical, or at least 99% phase Together）Heavy-chain variable domains.

In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people has and SEQ ID NO:Retouched in 18 The 17D20 stated（VH）It is substantially the same（For example, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, At least about 95%, or at least about 96% is identical, or at least about 97% identical, or at least about 98% is identical, or at least 99% identical）Weight Chain variable domains.In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people, which has, includes SEQ ID NO:18 or by SEQ ID NO:The heavy-chain variable domains of 18 compositions.

In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people has and SEQ ID NO:Retouched in 20 The 17D20_cd35VH2N11 stated（VH）It is substantially the same（For example, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96% is identical, and at least about 97% is identical, and at least about 98% is identical, or at least 99% phase Together）Heavy-chain variable domains.In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people have comprising SEQ ID NO:20 or by SEQ ID NO:The heavy-chain variable domains of 20 compositions.

In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people has and SEQ ID NO:Retouched in 21 The 17N16 stated（VH）It is substantially the same（For example, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, At least about 95%, or at least about 96% is identical, or at least about 97% identical, or at least about 98% is identical, or at least 99% identical）Weight Chain variable domains.In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people, which has, includes SEQ ID NO:21 or by SEQ ID NO:The heavy-chain variable domains of 20 compositions.

In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people has and any described in table 2 Light variable domains sequence is substantially the same（For example, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least About 90%, at least about 95%, or at least about 96% is identical, or at least about 97% identical, or at least about 98% is identical, or at least 99% phase Together）Light variable domains.

In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people has and SEQ ID NO:Retouched in 22 The 17D20 stated（VL）It is substantially the same（For example, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, At least about 95%, or at least about 96% is identical, or at least about 97% identical, or at least about 98% is identical, or at least 99% identical）It is light Chain variable domains.In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people, which has, includes SEQ ID NO:22 or by SEQ ID NO:The light chain of 22 compositions.

In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people has and SEQ ID NO:Retouched in 24 The 17D20_35VH-21N11VL stated（VL）It is substantially the same（For example, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% is identical, or at least about 97% identical, or at least about 98% is identical, or extremely Few 99% is identical）Light variable domains.In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody tool of theme people Have comprising SEQ ID NO:24 or by SEQ ID NO:The light chain of 24 compositions.

In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people has and SEQ ID NO:Retouched in 25 The 17N16 stated（VL）It is substantially the same（For example, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, At least about 95%, or at least about 96% is identical, or at least about 97% identical, or at least about 98% is identical, or at least 99% identical）It is light Chain variable domains.In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people, which has, includes SEQ ID NO:25 or by SEQ ID NO:The light chain of 25 compositions.

In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people has and SEQ ID NO:Retouched in 27 The 17N16_17N9 stated（VL）It is substantially the same（For example, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least About 90%, at least about 95%, or at least about 96% is identical, or at least about 97% identical, or at least about 98% is identical, or at least 99% phase Together）Light variable domains.In some embodiments, the anti-MASP-2 monoclonals inhibiting antibody of theme people have comprising SEQ ID NO:27 or by SEQ ID NO:The light chain of 27 compositions.

In some embodiments, anti-MASP-2 antibody of the invention includes weight or light chain, and it is by high stringent condition It is lower with encoding the nucleotide sequence coded of the nucleotide sequence hybridization of weight or light chain as described by table 2.High stringent condition bag Include at 50 DEG C or higher in 0.1xSSC（15mM salt solution/0.15mM sodium citrates）It is middle to be incubated.

In some embodiments, anti-MASP-2 inhibiting antibodies of the invention, which have, includes one or more CDR （CDR1, CDR2 and/or CDR3）Light chain variable district, the CDR compare Fig. 5 A or 5B in display or retouched in table 4 and table 5A-F The CDR for any light chain variable sequence stated amino acid sequence is substantially the same（For example, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% is identical, or at least about 97% identical, or at least about 98% It is identical, or at least 99% is identical）, or constitute including identical sequence or by identical sequence.

In some embodiments, anti-MASP-2 inhibiting antibodies of the invention, which have, includes one or more CDR （CDR1, CDR2 and/or CDR3）Light chain variable district, the CDR compare Fig. 5 A or 5B in display or retouched in table 4A-F and table 5 The CDR for any light chain variable sequence stated amino acid sequence is substantially the same（For example, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% is identical, or at least about 97% identical, or at least about 98% It is identical, or at least 99% is identical）, or constitute including identical sequence or by identical sequence.

Weight chain variable district

Table 3A：Heavy chain（Amino acid/11-20）

Table 3B：Heavy chain（Amino acid 21-40）

Table 3C：Heavy chain（Amino acid 41-60）

Table 3D：Heavy chain（Amino acid 61-80）

Table 3E：Heavy chain（Amino acid 81-100）

Table 3F：Heavy chain（Amino acid/11 01-118）

What is be illustrated below is the weight chain variable district of the female clone and son clone enumerated in table 2 above and table 3A-F（VH）Sequence Row.

Kabat CDR (31-35 (H1), 50-65 (H2) and 95-102 (H3)) are black matrix；And Chothia CDR (26-32 (H1), 52-56 (H2) and 95-101 (H3)) is underlined.

17D20 weight chain variable districts (VH) (SEQ ID NO:18):

17D20_35VH-21N11VL weight chain variable districts (VH) (SEQ ID NO:20):

17N16 weight chain variable districts (VH) (SEQ ID NO:21):

Table 4：Heavy chain CDR

Light chain variable district

Table 5A：Light chain（Amino acid/11-20）

Table 5B：Light chain（Amino acid 21-40）

Table 5C：Light chain（Amino acid 41-60）

Table 5D：Light chain（Amino acid 61-80）

Table 5E：Light chain（Amino acid 81-100）

Table 5F：Light chain（Amino acid/11 01-120）

What is be illustrated below is the light chain variable district of the female clone and son clone enumerated in table 2 above and table 5A-F（VL）Sequence Row.

Kabat CDR (24-34 (L1), 50-56 (L2) and 89-97 (L3)) are black matrix；And Chothia CDR (24-34 (L1), 50-56 (L2) and 89-97 (L3)) is underlined.No matter these areas pass through Kabat or Chothia systems Unified editing number is all identical.

17D20m light chain variable districts (VL) (SEQ ID NO:22):

17D20m_d3521N11 light chain variable districts (VL) (SEQ ID NO:24)

17N16m light chain variable districts (VL) (SEQ ID NO:25)

17N16m_d17N9 light chain variable districts (VL) (SEQ ID NO:27)

Table 6：Light chain CDR（Kabat/chothia）

On the one hand, the present invention provides the human monoclonal antibodies or its antigen-binding fragment for the separation for combining people MASP-2, Including：

（i）Include the weight chain variable district of CDR-H1, CDR-H2 and CDR-H3 sequence；With（ii）Including CDR-L1, CDR-L2 and CDR-L3 light chain variable district, wherein weight chain variable district CDR-H3 sequences include being described as SEQ ID NO:38 or SEQ ID NO: 90 amino acid sequence, and the modification of its conserved sequence, wherein light chain variable district CDR-L3 sequences include being described as SEQ ID NO: 51 or SEQ ID NO:94 amino acid sequence, and the modification of its conserved sequence, and the antibody of wherein described separation suppresses MASP- 2 complement activations relied on.

In one embodiment, the weight chain variable district CDR-H2 sequences include being described as SEQ ID NO:32 or 33 Amino acid sequence, and the modification of its conserved sequence.In one embodiment, the weight chain variable district CDR-H1 sequences include description For SEQ ID NO:28 or SEQ ID NO:29 amino acid sequence, and its conservative modification.In one embodiment, it is described light Chain variable region CDR-L2 sequences include being described as SEQ ID NO:93 amino acid sequence, and its conservative modification.In an implementation In scheme, the light chain variable district CDR-L1 sequences include being described as SEQ ID NO:91 or SEQ ID NO:92 amino acid sequence Row, and its conservative modification.In one embodiment, the CDR-H1 of the weight chain variable district includes SEQ ID NO：28.

In one embodiment, the CDR-H2 of the weight chain variable district includes SEQ ID NO：32.In an embodiment party In case, the CDR-H3 of the weight chain variable district includes SEQ ID NO：90（As shown in table 4）.In one embodiment, SEQ ID NO:Amino acid sequence described in 90 includes R in position 8（Arg）.

In one embodiment, the CDR-L1 of the light chain variable district includes SEQ ID NO：91（As shown in table 6）. In one embodiment, SEQ ID NO:Amino acid described in 91 includes E in position 2（Glu）.In an embodiment In, SEQ ID NO:Amino acid sequence described in 91 includes Y in position 8（Tyr）.

In one embodiment, the CDR-L2 of the light chain variable district includes SEQ ID NO：93（As shown in table 6）, And wherein SEQ ID NO:Amino acid sequence described in 93 includes K in position 2（Lys）.In one embodiment, SEQ ID NO:Amino acid sequence described in 93 includes Q in position 3（Gln）.

In one embodiment, the CDR-L3 of the light chain variable district includes SEQ ID NO：51.

In one embodiment, the antibody or its antigen-binding fragment combination people MASP-2, with 10nM or lower K_D.In one embodiment, the antibody or its antigen-binding fragment are surveyed in vitro is fixed in 1% human serum with 10nM Or lower IC₅₀Suppress C4 activation.In one embodiment, the antibody or its antigen-binding fragment are in 90% human serum With 30nM or lower IC₅₀Suppress C4 activation.In one embodiment, its conserved sequence modification is included or by such molecule Composition, the molecule include with described one or more variable domains identical variable regions, except in weight chain variable district In CDR region combination altogether 1,2,3,4,5,6,7,8,9 or 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and/or at most in the institute of light chain variable district State CDR region have combination altogether 1,2,3,4,5,6,7,8,9 or 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors.

On the other hand, the present invention provides the human antibody or its antigen-binding fragment for the separation for combining people MASP-2, wherein The antibody includes：I）a）Weight chain variable district, it includes i）Including SEQ ID NO：21 amino acid sequence 31-35 heavy chain CDR-H1；And ii）Including SEQ ID NO：21 amino acid sequence 50-65 heavy chain CDR-H2；And iii）Including SEQ ID NO：21 amino acid sequence 95-102 heavy chain CDR-H3；And b）Light chain variable district, it includes i）Including SEQ ID NO：25 or SEQ ID NO：27 amino acid sequence 24-34 light chain CDR-L1；And ii）Including SEQ ID NO：25 or SEQ ID NO：27 Amino acid sequence 50-56 light chain CDR-L2；And iii）Including SEQ ID NO：25 or SEQ ID NO：27 amino acid sequence Arrange 89-97 light chain CDR-L3；Or II）With the variable domains in its variant of other side identical, except described heavy At most combined in the CDR region of chain variable region altogether 1,2,3,4,5,6,7,8,9 or 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors and described At most combined in the CDR region of light chain variable district altogether 1,2,3,4,5,6,7,8,9 or 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, wherein institute State antibody or its variant suppresses the complement activation that MASP-2 is relied on.In one embodiment, the variant is one or more Include 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor selected from following position：The position 31 of the weight chain variable district, 32,33,34,35,51,52,53,54, 55th, 56,57,58,59,60,61,62,63,64,65,95,96,97,98,99,100 or 102.In one embodiment, institute State variant it is one or more selected from following position include 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor：The position 25 of the light chain variable district, 26,27, 29th, 31,32,33,51,52,89,92,93,95,96 or 97.In one embodiment, the heavy chain of the antibody includes SEQ ID NO:21.In one embodiment, the light chain of the antibody includes SEQ ID NO:25.In one embodiment, institute Stating the light chain of antibody includes SEQ ID NO:27.

On the other hand, the present invention provides the human monoclonal antibodies for the separation for combining people MASP-2, wherein the antibody bag Include：I）a）Weight chain variable district, it includes i）Including SEQ ID NO：20 amino acid sequence 31-35 heavy chain CDR-H1；And ii） Including SEQ ID NO：20 amino acid sequence 50-65 heavy chain CDR-H2；And iii）Including SEQ ID NO：18 or SEQ ID NO：20 amino acid sequence 95-102 heavy chain CDR-H3；And b）Light chain variable district, it includes i）Including SEQ ID NO：22 or SEQ ID NO：24 amino acid sequence 24-34 light chain CDR-L1；And ii）Including SEQ ID NO：22 or SEQ ID NO：24 Amino acid sequence 50-56 light chain CDR-L2；And iii）Including SEQ ID NO：22 or SEQ ID NO：24 amino acid sequence Arrange 89-97 light chain CDR-L3；Or II）With the variable domains in its variant of other side identical, except described heavy At most combined in the CDR region of chain variable region altogether 1,2,3,4,5,6,7,8,9 or 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors and described At most combined in the CDR region of light chain variable district altogether 1,2,3,4,5,6,7,8,9 or 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, wherein institute State antibody or its variant suppresses the complement activation that MASP-2 is relied on.In one embodiment, the variant is one or more Include 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor selected from following position：The position 31 of the weight chain variable district, 32,33,34,35,51,52,53,54, 55th, 56,57,58,59,60,61,62,63,64,65,95,96,97,98,99,100 or 102.In one embodiment, institute State variant it is one or more selected from following position include 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor：The position 25 of the light chain variable district, 26,27, 29th, 31,32,33,51,52,89,92,93,95,96 or 97.In one embodiment, the heavy chain of the antibody includes SEQ ID NO：20, or to SEQ ID NO：20 include at least 80%（For example, to SEQ ID NO：20 at least 85%, at least 90%, at least 95% or at least 98% homogeneity）Its variant of homogeneity.In one embodiment, the heavy chain of the antibody includes SEQ ID NO：18, or to SEQ ID NO：18 include at least 80%（For example, to SEQ ID NO：18 at least 85%, at least 90%, at least 95% Or at least 98% homogeneity）Its variant of homogeneity.In one embodiment, the light chain of the antibody includes SEQ ID NO：22, or to SEQ ID NO：22 include at least 80%（For example, to SEQ ID NO：22 at least 85%, at least 90%, at least 95% Or at least 98% homogeneity）Its variant of homogeneity.In one embodiment, the light chain of the antibody includes SEQ ID NO：24, or to SEQ ID NO：24 include at least 80%（For example, to SEQ ID NO：24 at least 85%, at least 90%, at least 95% Or at least 98% homogeneity）Its variant of homogeneity.

In one embodiment, the epitope in the CCP1 domains of the antibody binding MASP-2.

In one embodiment, the antibody binding people MASP-2, with 10nM or lower K_D.In an embodiment party In case, the antibody is surveyed in vitro to be fixed in 1% human serum with 10nM or lower IC₅₀Suppress C3b depositions.In an implementation In scheme, the antibody is in 90% human serum with 30nM or lower IC₅₀Suppress C3b depositions.

In one embodiment, the antibody is the antibody fragment selected from Fv, Fab, Fab', F (ab) 2 and F (ab') 2. In one embodiment, the antibody is single chain molecule.In one embodiment, the antibody is IgG2 molecules.One In individual embodiment, the antibody is IgG1 molecules.In one embodiment, the antibody is IgG4 molecules.In a reality Apply in scheme, the IgG4 molecules are mutated including S228P.

In one embodiment, the antibody does not suppress classical pathway substantially（That is, classical pathway activity at least 80%, or at least 90% or at least 95%, or at least 95% is complete）.

On the other hand, the present invention provides human monoclonal antibodies or its antigen-binding fragment of separation, and it is such as to pass through 10nM or lower K that surface plasma body resonant vibration is determined_DFrom people MASP-2 dissociation, and in the coated matrix of mannosan With 10nM or lower IC in 1% serum₅₀Suppress C4 activation.In some embodiments, the antibody or its antigen-binding fragment At least part of epitope recognized by reference antibody of specific recognition, wherein the reference antibody is included such as in SEQ ID NO: Weight chain variable district described in 20 and such as in SEQ ID NO:Light chain variable district described in 24, referring for example to antibody OMS646 （Referring to table 22）.According to foregoing, can be according to the antibody of the preferred embodiment of some the application or its antigen-binding fragment A kind of antibody or its antigen-binding fragment with any antibody competition combination people MASP-2 described herein, it was both（i）Specificity knot Antigen is closed, again（ii）Including VH and/or VL domains disclosed herein, or including CDR-H3 disclosed herein, or these antibody Any variant.Competition between combination member can be easily determined in vitro, such as using ELISA and/or by one Combination member adds specificity report molecule as label, and it can have one or more of the other untagged combination member In the case of be detected, so that the specific binding member with reference to same epitope or overlapping epitope can be identified.Therefore, it is current to provide Specific antibody or its antigen-binding fragment, including human antibody antigen binding site, itself and combination people MASP-2 described herein Antibody（Any one of OMS641-OMS646 for example as described in table 24）Competition binding people MASP-2.

The variant of MASP-2 inhibiting antibodies

Above-mentioned human monoclonal antibodies can be modified to provide the variant antibodies for the complement activation for suppressing MASP-2 dependences.Variant resists Body can be made by replacing, adding or lacking at least one amino acid of above-mentioned human monoclonal antibodies.Generally, these variants resist Body has the general features of above-mentioned human antibody, and includes the CDR of at least the above human antibody, or in certain embodiments, with The CDR of above-mentioned human antibody very similar CDR.

In preferred embodiments, the variant is included in one or more hypervariable regions of parental antibody one or many Individual 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.For example, the variant can include at least one in one or more CDR regions of parental antibody, such as From about one to about ten, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 substitutions, and preferably from about two to about six substitutions.In one embodiment, the variant is in one or more choosings Include 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor from following position：The position 31 of the weight chain variable district, 32,33,34,35,51,52,53,54,55, 56th, 57,58,59,60,61,62,63,64,65,95,96,97,98,99,100 or 102.In one embodiment, it is described to become Body it is one or more selected from following position include 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor：The position 25 of the light chain variable district, 26,27,29, 31st, 32,33,51,52,89,92,93,95,96 or 97.

In some embodiments, there are the variant antibodies variable domains with the theme antibody described in table 2 to exist Other side identical amino acid sequence, except at most combined in the CDR region of the weight chain variable district altogether 1,2, 3rd, 4,5 or 6 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors and/or at most combined in the CDR region of the light chain variable district altogether 1,2,3,4,5 Or 6 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, wherein the antibody or its variant suppress the complement activation that MASP-2 is relied on.

Generally, the variant will have has at least 75% amino acid with parental antibody weight or light variable domains sequence The amino acid sequence of sequence identity, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most Preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, at least 99% homogeneity.Homogeneity on this sequence Or homology is defined herein as in aligned sequences and introduces breach（If necessary）, to realize that maximized Percent sequence is same After property, with the percentage of parent antibody residues identical amino acid residue in candidate sequence.N-terminal, C-terminal or internal extension, Missing, or insertion antibody sequence（For example, such as signal peptide sequence, joint sequence or label, such as HIS labels）It should not be managed Solve as influence sequence identity or homology.Variant retains the ability that combines people MASP-2 and that preferably with than parental antibody The more excellent property of a little properties.For example, variant can have stronger binding affinity and/or enhanced suppression or blocking The ability for the complement activation that MASP-2 is relied on.

In order to analyze these properties, the Fab forms of variant and the Fab forms of parental antibody or the total length of variant should be compared The total length form of form and parental antibody, for example, due to having been found that the form of anti-MASP-2 antibody in biology disclosed herein Its activity is influenceed in determination of activity.This paper specific purpose variant antibodies are a kind of when the display at least about 10 compared with parental antibody Again, preferably at least about 20 times, and the antibody of most preferably at least about 50 times enhanced bioactivity.

The antibody of the present invention can be modified to strengthen desired property, and for example it can be that desirable control is anti- The serum half-life of body.Generally, complete antibody molecule has a very long serum continuation, and fragment（<60-80 kDa）Through Quickly filtered by kidney.Therefore, if it is desired to the long term of MASP-2 antibody, then MASP-2 antibody is preferably completely Total length IgG antibody（Such as IgG2 or IgG4）, and if it is desired to effect shorter MASP-2, then preferred antibody fragment.Strictly according to the facts Apply described in example 5, it has been determined that in the S228P substitution increase serum stabilities of IgG4 hinge areas.Therefore, in some embodiments, Theme MASP-2 antibody is the total length IgG4 antibody replaced with S228P.

Single-chain antibody

In one embodiment of the invention, MASP-2 inhibiting antibodies are single-chain antibodies, be defined as comprising light chain variable district, Weight chain variable district is connected as the molecule of the genetic modification of the single chain molecule of genetic fusion by suitable peptide linker.These are single-stranded anti- Body is also known as " scFv " or " scFv " antibody fragment.Generally, Fv polypeptides further comprise the polypeptide between VH and VL domains Joint, it, which enables scFv to form desired structure, is used for antigen binding.Can be with can with reference to MASP-2 scFv antibody The amino terminal in Bian Chong areas or the area's orientation that lightens of its carboxyl terminal.The exemplary scFv antibody of the present invention is described as SEQ ID NO:55-61 and SEQ ID NO: 66-68.

The method for producing antibody

In many embodiments, the nucleic acid of encoding schemes monoclonal antibody is introduced directly into host cell, and cell is being enough It is incubated under conditions of the coded antibody expression of induction.

In some embodiments, the present invention provides the anti-MASP-2 antibody of the coding present invention or the nucleic acid point of its fragment Antibody or its fragment described in son, such as table 2.In some embodiments, the present invention, which is provided, includes being selected from following nucleic acid The nucleic acid molecules of sequence：SEQ ID NO:19、SEQ ID NO:23、SEQ ID NO:26、SEQ ID NO:71、SEQ ID NO: 73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:97、SEQ ID NO:88 and SEQ ID NO:89.

In some embodiments, present invention offer includes the thin of the nucleic acid molecules of the anti-MASP-2 antibody of the coding present invention Born of the same parents.

In some embodiments, the present invention provides the table for the nucleic acid molecules for including the anti-MASP-2 antibody of the coding present invention Up to box.

In some embodiments, the present invention provides the method for producing anti-MASP-2 antibody, including culture includes code book The cell of the nucleic acid molecules of the anti-MASP-2 antibody of invention.

According to some related embodiments, there is provided the recombinant host including one or more constructs as described herein is thin Born of the same parents；Encode any antibody, CDR, VH or VL domain, or its antigen-binding fragment nucleic acid；Produce the side of coded product Method, methods described is so as to including the expression from code nucleic acid.Can be by cultivating the restructuring comprising nucleic acid under suitable condition Host cell conveniently realizes expression.By expressing after generation, any suitable technology separation and/or antibody purification can be used Or its antigen-binding fragment, and then use as desired.

For example, the cell of any expression for being suitable for expression cassette can be used as host cell, for example, yeast, insect, The cells such as plant.In many embodiments, using the mammalian host cell line for not producing antibody generally, in fact for example Under：Monkey kidney cell（COS cells）, the dirty CVI cells of monkey kidney that are converted by SV40（COS-7, ATCC CRL 165 1）；People's embryo Nephrocyte（HEK-293, Graham etc.,J. Gen Virol. 36:59 (1977)）；Baby hamster kidney cell（BHK, ATCC CCL 10）；Chinese hamster ovary cell（CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. (USA) 77: 4216, (1980)）；Properties of Sertoli Cells Isolated from Mice Testis（TM4, Mather,Biol. Reprod. 23:243-251 (1980)）； MK cells（CVI ATCC CCL 70）；African green monkey kidney cell（VERO-76, ATCC CRL-1587）；Human cervical carcinoma cell （HELA, ATCC CCL 2）；MDCK（MDCK, ATCC CCL 34）；Buffalo rats liver（BRL 3A, ATCC CRL 1442）；Human pneumonocyte（W138, ATCC CCL 75）；Human liver cell（Hep G2, HB 8065）；Mouse mammary tumor（MMT 060562, ATCC CCL 51）；TRI cells（Mather etc.,Annals N.Y. Acad. Sci 383:44-68 (1982)）； NIH/3T3 cells（ATCC CRL-1658）And mouse Lcell（ATCC CCL-1）.For those skilled in the art, additionally Cell line will become apparent.Various cell lines can be from American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 are obtained.

It is well known in the art by the method for nucleic acid into cells.Suitable method includes electricity conversion, gene gun technology （particle gun technology）, calcium phosphate precipitation, direct microinjection etc..The selection of method, which is generally depended on, to be converted Cell type and conversion occur environment（That is, it is external, ex vivo or internal）.The general discussion of these methods can be with Ausubel is seen, etc.,Short Protocols in Molecular Biology, 3d ed., Wiley & Sons, 1995.In some embodiments, the gene transfer technique mediated using liposome and calcium.

Theme nucleic acid has been imported after cell, and cell is typically incubated, generally at 37 DEG C, sometimes under selection, and it is suitable to carry out Time to allow antibody expression.In most of embodiments, antibody is typically secreted into the culture that cell grows wherein The supernatant of base.

In mammalian host cell, it is possible to use some expression systems based on virus are to express theme antibody. In the case that adenovirus is used as expression vector, the antibody of coding aim sequence may be coupled to adenovirus transcription/translation control again Compound, such as late promoter and triplet targeting sequencing.This mosaic gene and then can be recombinated by vitro and in vivo and inserted Adenoviral gene group.The insertion of viral genome nonessential region（Such as E1 or E3 areas）It will obtain vibrant and can infect Host in expression antibody molecule recombinant virus.(see, e.g. Logan ＆ Shenk,Proc. Natl. Acad. Sci. USA 81:355-359 (1984)).Expression efficiency can be by including appropriate transcription enhancer element, tanscription termination Son etc. is strengthened（Referring to Bittner etc.,Methods in Enzymol. 153:51-544 (1987)）.

Produced for the long-term of recombinant antibodies, high yield, stable expression can be used.For example, stable table can be transformed Up to the cell line of antibody molecule.Host cell can be converted with immunoglobulin expression box and selectable mark, rather than be made With the expression vector originated comprising virus replication.Import after exogenous DNA, the cell of transformation can be allowed raw in enriched medium It is long 1-2 days, and then go to selective medium.Optional mark in recombinant plasmid assigns selection resistance, and allows Cytotostatic by plasmid integration to chromosome and grow form colony, then it can be cloned and be extended to cell line.Should The cell line of transformation can be particularly used in screening and evaluate the compound directly or indirectly interacted with antibody molecule.

Once the antibody molecule of the present invention has been produced, it can be by any known in the art for purifying immune globulin The method of white molecule, for example, pass through chromatography（For example, ion exchange, affinity, especially by the specific antigen after albumin A Affinity, and size exclusion column chromatography（sizing column chromatography））, centrifugation, dissolving sex differernce, or pass through Any other standard technique for purifying protein is purified.In many embodiments, antibody is from cell is secreted into culture medium And harvested from culture medium.For example, can include the nucleotide sequence of encoded signal peptide in the neighbouring of the code area of antibody or fragment, For example it is provided in SEQ ID NO:71 nucleotides 1-57, coding such as SEQ ID NO:The signal provided in 72 amino acid/11-19 Peptide.The signal peptide can be incorporated into the 5' ends of the amino acid sequence described herein of neighbouring theme antibody, with Help Topic antibody Generation.

Pharmaceutical carriers and delivery vehicle

On the other hand, the present invention is provided to the composition of the ill effect for the complement activation for suppressing MASP-2 dependences, described group Compound includes the anti-MASP-2 inhibiting antibodies of people and pharmaceutically acceptable carrier of therapeutically effective amount.

Generally, people's MASP-2 inhibiting antibodies composition of the invention that combination has the therapeutic agent of any other selection is adapted to Included in pharmaceutically acceptable carrier.Carrier is nontoxic, bio-compatible and is selection so as not to can adverse effect MASP-2 inhibiting antibodies（Any other therapeutic agent wherein combined）Biological activity.Exemplary pharmacy for polypeptide Acceptable carrier is described in the U.S. Patent number 5,211,657 for authorizing Yamada.Anti- MASP-2 antibody can be configured to solid, Semisolid, gel, the prepared product of liquid or gas form, such as tablet, capsule, pulvis, granule, ointment, solution, Suppository (depositories), inhalant and injection, are applied for oral, parenteral or surgical operation.Present invention additionally comprises logical Cross composition being coated on medical treatment device and carry out local application etc..

Suitable carrier for the parenteral delivery via injection, infusion or flushing and local delivery includes distilled water, phosphorus Acid buffering physiological saline, standard ringer's solution or Lactated Ringer liquid, glucose solution, HankShi solution or propane diols.This Outside, sterile expressed oi can be used as solvent or suspension media., can be using any biocompatibility oil for this purpose, including close Into monoglyceride or diglyceride.In addition, aliphatic acid (such as oleic acid) can be used in the preparation of injection.Can be by carrier and examination Agent, which is prepared, turns into liquid preparation, supensoid agent, polymerizable or not polymerizable gel, paste or ointment.

Carrier may also comprise delivery vehicle so that the delivering of one or more reagents continues (to extend, delay or adjust Section), or strengthen delivering, absorption, stability or the Pharmacokinetic Characteristics of one or more therapeutic agents.This delivery vector can Including by way of non-limiting example：By protein, liposome, carbohydrate, anthropogenics, inorganic compound, poly- Particulate, microballoon, nanosphere, the nanoparticle of Heshui gel, copolymer hydrogel and polymer micelle composition.Suitable hydrogel and glue Beam delivery system includes the PEO disclosed in the A2 of WO 2004/009664:PHB:PEO copolymers and copolymer/cyclodextrin complexes And PEO the and PEO/ cyclodextrin complexes disclosed in the A1 of U.S. Patent Application Publication No. 2002/0019369.These hydrogels Can local injection to set action position, or subcutaneously or intramuscularly inject to form slow-release depot（depot）.

Pass medicine for intra-articular, MASP-2 inhibiting antibodies can be loaded on above-mentioned injectable liquid or gel carrier, In the sustained release drug delivery medium or hyaluronic acid or derivatives of hyaluronic acids of above-mentioned injectable.

(ICV) passs medicine, suitable aseptic delivery system (such as liquid preparation in intrathecal (IT) or the ventricles of the brain；It is gel, mixed Suspension etc.) it can be used to using medicine of the invention.

The composition of the present invention may also include biocompatible excipient, such as dispersant or wetting agent, suspending agent, dilution Agent, buffer, penetration enhancer, emulsifying agent, adhesive, thickener, flavouring (being used to orally administer).

In order to obtain the anti-MASP-2 antibody of the high concentration for local delivery, antibody can be formulated as in the solution heavy Form sediment or the suspension of crystal is used for follow-up injection, such as intramuscular injection for reservoir.

As for anti-MASP-2 antibody, more specifically, can by the compound of injection dosage solution or supensoid agent Parenterally exemplary formulation, the compound is in physiologically acceptable diluent and pharmaceutical carrier, pharmaceutical carrier Can be sterile liquid, such as water, oil, salt solution, glycerine or ethanol.In addition, can be deposited in the composition comprising anti-MASP-2 antibody In the auxiliary substance such as wetting agent or emulsifying agent, surfactant, pH buffer substance.The added ingredient of pharmaceutical composition includes Crude oil（petroleum）(such as animal, plant or synthesis source), such as soybean oil and mineral oil.In general, dihydric alcohol Such as propane diols or polyethylene glycol are the liquid-carriers of preferred injection solution.

Can also with depot injection or implantation prepared product in the form of apply anti-MASP-2 antibody, the preparation can by allow live The mode of property agent sustained release or pulse release is prepared.

Can apply in many ways includes the pharmaceutical composition of MASP-2 inhibiting antibodies, and this is depended on topically or systemically Whether mode of administration is most suitable for disease to be treated.In addition, it is herein above described on external Reperfu- sion method, can be by inciting somebody to action The composition of the present invention introduces recycling blood or blood plasma to apply MASP-2 inhibiting antibodies.Moreover, the composition of the present invention Can by by composition be coated with mix above implantable medical treatment device or the inside and deliver.

Systemic delivery

Term " systemic delivery " as used herein and " systemic administration " expected including but not limited to oral and parenteral outer approach, Including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, suction, sublingual, oral cavity, part, percutaneous, intranasal, rectum, vagina and Other route of administration, the antibody delivered is effectively dispersed to be expected one or more positions of therapeutic action by them.For The optimization approach of the systemic delivery of this composition includes intravenous, intramuscular, subcutaneous and suction.It should be appreciated that for The selected reagent in concrete composition of the present invention, definite systemic administration approach will partly consider reagent pair with it is specific The sensitiveness of the related metabolic conversion approach of route of administration is determined.

MASP-2 inhibiting antibodies and polypeptide can be delivered to by any suitable method to be needed in its subject.Pass Send MASP-2 antibody and polypeptide method include by oral, lung, it is parenteral (such as intramuscular, intraperitoneal, intravenous (IV) or Be subcutaneously injected), suck (such as via attritive powder preparation), percutaneous, intranasal, vagina, rectum or sublingual administration route, and can It is configured to the formulation suitable for respective method of administration.

For example, can be by the way that MASP-2 suppression antibody and peptide be applied on the body film that can absorb polypeptide, example Such as nose film, gastrointestinal membranes and bung skin, and be introduced into vivo.Generally polypeptide and penetration enhancer are applied to together absorbable On film.(see, e.g. Lee, V.H.L.,Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, V.H.L., J. Controlled Release13:213, 1990; Lee, V.H.L., Ed., Peptide and Protein Drug Delivery,Marcel Dekker, New York (1991);DeBoer, A.G., etc.,J. Controlled Release 13:241, 1990.) for example, STDHF is the synthesis of derivatives of fusidinic acid, it is class in structure The steroid surfactant of cholate is similar to, and has been used as the penetration enhancer that intranasal passs medicine.(Lee, W.A.,Biopharm.22, Nov./Dec. 1990。)

The MASP-2 inhibiting antibodies and polypeptide associated with another molecule (such as lipid) can be introduced, to protect polypeptide not by enzyme Degraded.For example, covalently bound polymer, especially polyethylene glycol (PEG) have been used to protect some protein not internal Enzyme hydrolysis, so as to extend half-life period (Fuertges, F. etc., J. Controlled Release 11:139,1990). Through reporting many polymer system (Bae, Y.H. etc., J. Controlled Release 9 for protein delivery: 271,1989；Hori, R. etc., Pharm. Res. 6:813,1989；Yamakawa, I. etc., J. Pharm. Sci. 79: 505,1990；Yoshihiro, I. etc., J. Controlled Release 10:195,1989；Asano, M. etc., J. Controlled Release 9:111,1989；Rosenblatt, J. etc., J. Controlled Release 9:195, 1989；Makino, K., J. Controlled Release 12:235,1990；Takakura, Y. etc., J. Pharm. Sci. 78:117,1989；Takakura, Y. etc., J. Pharm. Sci. 78:219,1989).

Recently, serum stability has been developed and circulating half-life obtains improved liposome (see, for example, Webb U.S. State's patent No. 5,741,516).Moreover, to the various methods of liposome and liposome sample prepared product as possible pharmaceutical carrier It is reviewed (see, for example, Szoka U.S. Patent number 5,567,434；Yagi U.S. Patent number 5,552,157； Nakamori U.S. Patent number 5,565,213；Shinkarenko U.S. Patent number 5,738,868 and the Gao U.S. are special Profit number is 5,795,587).

For percutaneous application, MASP-2 can be suppressed antibody and polypeptide and other suitable composition (such as carriers and/or assistant Agent) combination.Property to these other compositions is not limited, and must be pharmaceutically acceptable for being applied only for its expection , and the activity of active component in composition can not be reduced.The example of suitable carrier is included with or without the soft of collagen purification Cream, emulsifiable paste, gel or suspension.MASP-2, which suppresses antibody and polypeptide, can also be impregnated into transdermal patch, plaster and bandage, excellent Select liquid or semi-liquid form.

In the periodic basis at the interval that can be determined in the level for needed for maintaining treatment effect, this hair of systemic administration Bright composition.For example, composition (such as subcutaneous injection) can be applied by every 2-4 weeks or with more low-frequency interval.Give Prescription case will be considered the various factors acted on associated with reagent may be influenceed to determine by doctor.These factors will include to be treated Progress extent, patient age, sex and the body weight and other clinical factors of situation.It will make for the dosage of each indivedual reagents For the MASP-2 inhibiting antibodies included in composition and any medicine delivery medium (such as sustained release delivery medium) Change in the presence of the function with property.In addition, can be dynamic in the medicine generation for considering frequency of administration and the one or more reagents delivered Dosage is adjusted after the change of mechanical behavior frequency.

Local delivery

As used herein, term " part " includes application medicine on expected local action position or around it, and can wrap Include and be for example delivered locally to skin or other affected tissues, eye pass medicine, intrathecal (IT), (ICV), intra-articular, intracavitary, cranium in the ventricles of the brain Apply, dispose or rinse in interior or alveolar.The local application of relatively low-dose can preferably be applied to avoid systemic side effects, And precisely control the surfactant concentration of Delivery time and local delivery site.No matter in metabolism, blood between patient Change in terms of stream, local application all provides concentration known in target site.Also dosage is controlled by direct modes of delivery To improvement.

The local delivery of MASP-2 inhibiting antibodies can be in the mistake with operation method treatment disease or the surgical operation of situation Realized in journey, such as in such as arterial bypass, atherectomy, laser surgey, ultrasonic operation, sacculus blood During Tuboplasty and Stent Implantation etc. are performed the operation.Applied for example, can be combined MASP-2 inhibitor with balloon angioplasty Use subject.Balloon angioplasty includes that the conduit insertion intra-arterial for being vented sacculus will be connected with.Sacculus will be vented to put Near atherosclerotic plaque, and to airbag aeration patch is extruded to vascular wall.As a result, balloon surface and blood vessel table The vascular endothelial cell layer contact in face.Can be in the way of allowing reagent to be discharged at atherosclerotic plaque position, by MASP- 2 inhibiting antibodies are attached on balloon angioplasty catheter.Reagent can be attached to ball according to standard method known in the art On ductus bursae.For example, can be stored in reagent in the compartment of foley's tube until inflated, now medicine is released to part In environment.Or, can be by reagent-impregnated in balloon surface, so that when inflated, reagent just touches the cell of arterial wall. Also can in porous foley's tube delivery of agents, see, for example, Flugelman, M.Y. etc., Circulation 85:1110- 1117,1992.It see also in published PCT application WO 95/23161 for treatment albumen is attached into balloon angioplasty The supravasal illustrative methods of art.Similarly, MASP-2 inhibiting antibodies can be also wrapped into applied to the gel on support or poly- Close coating material in, or can by MASP-2 inhibiting antibodies mix timbering material in so that support blood vessel placement after just MASP-2 inhibiting antibodies are eluted out.

Therapeutic scheme

MASP-2 inhibiting antibodies composition for treatment of arthritis and other musculoskeletal diseases can pass through intra-articular injection Carry out local delivery.These compositions can suitably include sustained release delivery carrier.As may wherein need the another of local delivery Individual example, for treat the MASP-2 inhibiting antibodies composition of disease of the genitourinary system can suitably instill in bladder or In other urogenital structures.

In prophylactic applications, described pharmaceutical composition be enough to eliminate or reduce condition symptoms occurrence risk amount apply It is susceptible or there is the subject of risk in addition in the related situation of the complement activation that relies on MASP-2.In treatment use, Described pharmaceutical composition be enough to alleviate or at least partly reduce condition symptoms therapeutically effective amount be applied to suspection or already The subject of situation with the MASP-2 complement activation correlations relied on.In prevention and treatment scheme, including MASP-2 suppresses Property antibody composition can be applied with several dosage, until obtain enough treatment results in subject.The present invention's The application of MASP-2 inhibiting antibody compositions can be carried out by the single administration of composition, or limited order of administration, be used In the treatment of acute condition, such as reperfusion injury or other wounds.Or, can at regular intervals it be applied in longer period of time With composition, for treating chronic condition, such as arthritis or psoriasis.

The MASP-2 inhibitions composition used in the present invention can be after the acute events for causing lectin pathway to activate Deliver immediately or quickly, such as after ischemic events and the Reperfu- sion of ischemic tissue.Example includes myocardial ischemia-reperfusion, kidney Dirty ischemia-reperfusion, cerebrum ischemia Reperfu- sion, organ transplant and refer to toe/limbs and adhere to again.Other acute examples include septicemia. The MASP-2 inhibitions composition of the present invention can as early as possible be applied after the acute events of activation lectin pathway, preferably 12 In hour, and more preferably for example passed within the 2-3 hours of trigger event by the whole body of MASP-2 inhibition compositions Send.

The method and composition of the present invention can be used for that suppression is typically due to diagnosis and curative drug and operation method are led The inflammation and correlated process of cause.In order to suppress these processes, MASP-2 inhibitions composition of the invention can be answered with peri-operation period With.As used herein " peri-operation period " refer to inhibition composition operation consent and/or operation in and/or Post operation apply, that is, exist Before operation, before and during operation, perioperatively, operation consent, during and after, during operation, during and after operation, or Post operation.Peri-operation period application can be by operation（surgical）Or operation（procedural）Described in the local application of position Composition carry out, for example by injection or position accomplished continuously or intermittently perfusion or pass through systemic administration.For MASP-2 inhibitions The suitable method of the local peri-operation period delivering of antibody-solutions is disclosed in the U.S. Patent number 6,420 for authorizing Demopulos, 432 and authorize Demopulos U.S. Patent number 6,645,168.For the cartilage protection group including MASP-2 inhibiting antibodies The suitable method of the local delivery of compound is disclosed in the A2 of international PCT patent application WO 01/07067.For including MASP-2 The suitable method and composition of the targeting systemic delivery of the cartilage protection composition of inhibiting antibody is disclosed in international PCT patent Apply for the A2 of WO 03/063799.

Dosage

MASP-2 inhibiting antibodies can be applied to treatment effective dose needs its subject, to treat or alleviate MASP-2 The related situation of the complement activation of dependence.The amount that treatment effective dose refers to MASP-2 inhibiting antibodies is enough to cause the symptom of situation Alleviate.

The toxicity and treatment effect of MASP-2 inhibiting antibodies can be determined by the method for pharmacy of standard, dynamic using experiment Thing model, such as cercopithecus aethiops, it is as described herein.Using these animal models, standard method can be used to determine NOAEL（Nothing The ill effect level of observation）And MED（Minimum level of significance）.The dose ratio of NOAEL and MED effects is treatment ratio, its It is expressed as NOAEL/MED ratios.Show that the MASP-2 inhibiting antibodies of big treatment ratio or index are most preferred.From cell training The data for supporting measure and zooscopy acquisition can be used for preparing a series of dosage using in people.MASP-2 inhibiting antibodies Dosage be preferably in a series of circulation compositions including MED, with seldom or avirulent.Depending on the dosage used With the route of administration utilized, dosage can change within this range.

For any compound preparation, animal model can be used to estimate treatment effective dose.For example, can be in animal mould Dosage is prepared in type to obtain the circulating plasma concentration range including MED.MASP-2 inhibiting antibodies can also be measured in blood plasma In quantitative level, for example pass through high performance liquid chroma- tography.

In addition to toxicity research, the amount of MASP-2 albumen present in subject living and MASP-2 suppressions are also based on The binding affinity estimation effective dose of property antibody processed.Show that MASP-2 levels are with 500 ng/ml models in normal volunteer The MASP-2 levels that the low-level enclosed is present in serum, and particular subject can use the quantitative determination for MASP-2 To determine, the quantitative determination is described in Moller-Kristensen M., et al.,J. Immunol. Methods 282:159-167,2003, it is incorporated herein by reference herein.

Generally, including MASP-2 inhibiting antibodies the composition applied dosage depend on following factors and change： Subject age, body weight, height, sex, general curative situation and prior medical history.Illustratively, MASP-2 inhibiting antibodies Can be with from about 0.010-10.0mg/kg, preferably 0.010-1.0mg/kg, more preferably 0.010-0.1mg/kg subject's body weight Dosage range is applied.

Treatment effect of the MASP-2 inhibitions composition and method of the present invention in given subject, and appropriate dosage It can be determined to determine according to complement well known to those skilled in the art.Complement generates many specific products.For major part These activation products, including small activation segment C3a, C4a and C5a and big activation segment iC3b, C4d, Bb and sC5b9, It is past during the decade, developed sensitivity and it is specific measure and be commercially available.These most of surveys It is fixed using with fragment rather than they formed from native protein on exposure neoantigen（neoantigen）The antibody of reaction, Determine these very simple and special.It is most of to rely on elisa technique, although still sometimes using spoke for C3a and C5a The immunoassays of penetrating property.These later measure measure untreated fragment and their " desArg " fragments, and it is discovery in circulation Principal mode.Untreated fragment and C5a_desArgQuickly removed and so as to very low by combining cell surface receptor Concentration exist, and C3a_desArgCell is not combined and is accumulated in blood plasma.C3a measurement there is provided complement activation it is sensitive, The indicant that approach is relied on.It can be activated by measuring Bb Segment evaluations alternative route.Film attacks the fluid phase of pathway activation （fluid-phase）Product, sC5b9 detection provides the evidence that complement activation is completed.Because agglutinin and classical pathway generation Same activation products, C4a and C4d, the measurement of the two fragments do not provide any on which the generation activation of the two approach The information of product.

The suppression for the complement activation that MASP-2 is relied on is characterized as at least one following change of complement system composition, its conduct Occur using the result of the anti-MASP-2 antibody according to the present invention：MASP-2 rely on complement activation system product C4b, C3a, C5a and/or C5b9（MAC）Generation or generation suppression, C4 cutting and C4b deposit reduction, or C3 cutting and C3b deposit Reduce.

Product

On the other hand, the present invention provides the manufacture article comprising people MASP-2 inhibiting antibodies or its antigen-binding fragment, such as It is described herein to be suitable for treating the unit dosage form applied to people experimenter, for example, such as the unit in 1mg-5000mg scopes Dosage, such as 1mg-2000mg, such as 1mg-1000mg, such as 5mg, 10mg, 50mg, 100mg, 200mg, 500mg or 1000mg.In some embodiments, the product include container on container or related label or package insert.Properly Container include, for example, bottle, bottle, syringe etc..The container can be formed from various materials such as glass or plastics.Hold Device accommodates the composition effective to treatment situation, it is possible to sterile gateway（For example, container can be intravenous solution bag Or the bottle with the plug that can be run through by hypodermic needle）.At least one of composition activating agent is the MASP-2 of the present invention Inhibiting antibody or its antigen-binding fragment.Label or package insert indicate that the composition is used to treat particular condition.Mark Label or package insert will further comprise the explanation to patient's administration of antibodies composition.It is also contemplated that as described herein including combination The product and kit for the treatment of.

The therapeutical uses of anti-MASP-2 inhibiting antibodies

On the other hand, the present invention provide in people experimenter suppress MASP-2 rely on complement activation method, including with The amount for suppressing the complement activation that MASP-2 is relied in the people experimenter enough applies the anti-MASP-2 suppressions of human monoclonal of the present invention Property antibody processed.

According to this aspect of the invention, as described in example 10 above, anti-MASP-2 inhibiting antibodies of the invention are in vein Lectin pathway in cercopithecus aethiops can be suppressed after interior administration.Such as table 24, shown in embodiment 8, find what is used in this research Antibody OMS646 is more effective in human serum.As is known in the art, non-human primate is commonly used as evaluating The model of Antybody therapy.

Such as in U.S. Patent number 7,919,094, Co-pending U.S. Patent Application series number 13/083,441 and common Pending U.S. Patent Application Serial 12/905,972（Each of which distributes to Omeros Corporation, the application's Assignee）Described in, each of which is incorporated by reference into, and it is many acute that the complement activation that MASP-2 is relied on has been indicated as promotion With the pathogenesis of chronic disease states, including the vascular condition of complement-mediated, ischemical reperfusion injury, dynamic that MASP-2 is relied on Pulse atherosclerosis, inflammatory bowel road illness, lung condition, external refilling process, skeletal muscle situation, kidney shape condition, skin, Organ or tissue's transplanting, neurological conditions or damage, blood disorder, urogenital tract situation, diabetes, chemotherapy or radiation are controlled Treatment, malignant tumour, endocrine disturbance, blood coagulation disorders, or ophthalmic conditions.Therefore, MASP-2 inhibiting antibodies of the invention can be with For treating above-mentioned disease and situation.

It is further described that the MASP-2 inhibiting antibodies of the present invention in treatment there is acute radiation to integrate in such as embodiment 11 Levy the risk of adverse effect or the mammalian subject that is adversely affected by acute radiation syndrome in be effective, thus demonstrate,prove Treatment effect in phaneroplasm.

The following example only explains the optimal mode for being presently contemplated for the practice present invention, but should not be construed as limiting this hair It is bright.

Embodiment

Embodiment 1

Derive polypeptide and catalytically inactive present embodiment describes recombinant full-lenght people, rat and mouse MASP-2, MASP-2 The recombination expression and albumen of MASP-2 mutant forms are produced.

Total length people and rat MASP-2 expression：

Encoding human MASP-2 polypeptides had into targeting sequencing（SEQ ID NO:2）People's MASP-2 full length cDNA sequences (SEQ ID NO:1) it is subcloned into mammalian expression vector pCI-Neo (Promega), it is in cmv enhancer/promoter region control The lower driving eukaryotic expression of system (is described in Kaufman R.J. etc., Nucleic Acids Research 19:4485-90,1991； Kaufman, Methods in Enzymology, 185:537-66 (1991)).By having for encoding rat MASP-2 polypeptides Targeting sequencing（SEQ ID NO:5）Rat MASP-2 full-length cDNAs (SEQ ID NO:4) it is subcloned into pED expression vectors. Then standard calcium phosphate transfection method (being described in Maniatis etc., 1989) is used, MASP-2 expression vectors are transfected into adherent Chinese hamster ovary line DXB1 in.The cell growth transfected with these constructs obtains very slow, shows coded Protease has cytotoxicity.The mature form of people's MASP-2 albumen（SEQ ID NO:3）With the ripe shape of rat MASP-2 albumen Formula（SEQ ID NO:6）It is secreted into culture medium, and is separated as following.

The MASP-2 of total length catalytically inactive expression：

General principle：

In identification subcomponent MBL, c-type agglutinin CL-11 or fiber gelatinized protein (the fiber gelatinized egg of L- fiber gelatinized proteins, H- White or M- fiber gelatinized proteins)（It is referred to as agglutinin）After their own carbohydrate pattern, MASP-2 passes through self-catalysis Cutter activation.Cause MASP-2 activate autocatalytic cleavage frequently occur in from serum separation MASP-2 during, Huo Zhe During purifying after recombination expression.It is used as antigen to obtain more stable protein prepared product, is replaced by using alanine residue Serine residue present in protease domain catalytic triads, produces the MASP-2, referred to as MASP- of catalytically inactive form 2A, (the SEQ ID NO in ripe rat MASP-2 albumen:6 Ser617 to Ala617)；Or into acquaintance's MASP-2 albumen (SEQ ID NO:3 Ser618 to Ala618).

To generate people and the rat MASP-2A albumen of catalytically inactive, US2007/0172483 is used（It is incorporated by reference into Herein）Described in carry out direct mutagenesis.PCR primer is purified after prepared by agarose gel electrophoresis and band, and uses standard It is overlapping that tailing method produces simple gland glycosides.Then the MASP-2A for adding adenosine tail is cloned into pGEM-T easy carriers, then converted Into Escherichia coli.Further by people and rat MASP-2A be each further subcloned into mammalian expression vector pED or PCI-Neo is simultaneously transfected into Chinese hamster ovary line DXB1 as described below.

The structure of the expression plasmid of peptide zone comprising derived from human MASP-2

Using MASP-2 signal peptides (SEQ ID NO:2 residue 1-15) it is different to secrete to produce following construct MASP-2 domains.Pass through PCR amplification codings MASP-2 (SEQ ID NO:3) residue 1-121 region is (equivalent to N-terminal CUB1 Domain) express people MASP-2 CUBI domains (SEQ ID NO to prepare:7) construct.Pass through PCR amplification codings MASP-2 (SEQ ID NO:3) people MASP- is expressed in residue 1-166 region (equivalent to N-terminal CUB1/EGF domains) to prepare 2 CUBI/EGF domains (SEQ ID NO:8) construct.Pass through PCR amplification codings MASP-2 (SEQ ID NO:3) residue Tied to prepare expression people MASP-2 CUBI/EGF/CUBII in 1-277 region (equivalent to N-terminal CUB1/EGF/CUBII domains) Structure domain (SEQ ID NO:9) construct.Pass through PCR amplification codings MASP-2 (SEQ ID NO:3) residue 122-166 area People MASP-2 EGF domains (SEQ ID NO are expressed to prepare in domain (equivalent to EGF domains):10) construct.Pass through PCR Amplification coding MASP-2 (SEQ ID NO:3) residue 278-671 region (equivalent to CCPI/CCPII/SP domains) is made Standby expression people MASP-2 CCPI/CCPII/SP domains (SEQ ID NO:11) construct.Pass through PCR amplification codings MASP- 2 (SEQ ID NO:3) people MASP-2 is expressed in residue 278-429 region (equivalent to CCPI/CCPII domains) to prepare CCPI/CCPII domains (SEQ ID NO:12) construct.Pass through PCR amplification codings MASP-2 (SEQ ID NO:3) it is residual MASP-2 CCPI domains (SEQ ID NO are expressed to prepare in base 278-347 region (equivalent to CCPI domains):13) Construct.Pass through PCR amplification codings MASP-2 (SEQ ID NO:3) residue 348-671 region is (equivalent to CCPII/SP knots Structure domain) express MASP-2 CCPII/SP domains (SEQ ID NO to prepare:14) construct.Pass through PCR amplification codings MASP-2 (SEQ ID NO:3) MASP-2 is expressed in residue 348-429 region (equivalent to CCPII domains) to prepare CCPII domains (SEQ ID NO:15) construct.Pass through PCR amplification codings MASP-2 (SEQ ID NO:3) residue 429- MASP-2 SP domains (SEQ ID NO are expressed to prepare in 671 region (equivalent to SP domains):16) construct.

Use Vent_RPolymerase and pBS-MASP-2 is as template, according to the PCR method being established above, is expanded by PCR Above mentioned MASP-2 domains.The 5' primer sequences of sense primer are introduced at the 5' ends of PCR primerBamHI restriction sites （Underscore）.The antisense primer of each MASP-2 domains is designed as introducing terminator codon simultaneously in the end of each PCR primer It is followed byEcoRI sites.Once amplification, DNA fragmentation is usedBamHI andEcoRI digests and is cloned into the corresponding of pFastBac1 carriers Site.Mapped by restricted digestion and characterize obtained construct, and confirmation is sequenced by dsDNA.

MASP-2 recombinant eukaryon expression, and the rat of non-enzymatic activity and people MASP-2A albumen are produced.

Above-mentioned MASP-2 and MASP-2A expression constructs use standard calcium phosphate infection protocol（Maniatis etc., 1989） It is transfected into DXB1 cells.MASP-2A is produced in serum free medium to ensure that prepared product is not polluted by other haemocyanins.Often Second day from converging cell harvesting culture medium（Four times altogether）.For two species each, MASP-2A level is recombinated averagely about 1.5mg/ rises culture medium.

MASP-2A protein purifications：MASP-2A（Above-mentioned Ser-Ala mutant）By affinity chromatography on MBP agarose columns Purifying.This strategy can without using external label fast purifying.MASP-2A（Use isometric sample-loading buffer（50 mM Tris-Cl, pH 7.5, includes 150 mM NaCl and 25 mM CaCl₂）The 100-200ml culture mediums of dilution）It is loaded into use The MBP agarose affinity columns of 10ml sample-loading buffer pre-equilibrations（4ml）On.After being cleaned with other 10ml sample-loading buffers, albumen Use 50mM TrisCl（PH7.5, includes 1.25M NaCl and 10mM EDTA）It is eluted in 1ml fractions.Level comprising MASP-2A Divide and identified by SDS- polyacrylamide gel electrophoresises.If necessary, MASP-2A by ion-exchange chromatography in MonoQ posts（HR5/ 5）On be further purified.Albumen NaCl containing 50mM 50mM TrisCl, pH7.5 dialyse and are loaded into same buffer solution and put down On the post of weighing apparatus.After cleaning, with reference to MASP-2A 10ml 0.05-1M NaCl gradient elutions.

As a result：0.25-0.5mgMASP-2A protein yields are obtained from 200ml culture mediums.Due to glycosylation, pass through The molecular weight for the 77.5kDa that MALDIMS is determined is more than the calculated value of unmodified polypeptide（73.5kDa）.Observed quality is Due to the glycan attachment at each N glycosylation sites.MASP-2A is migrated on SDS- polyacrylamide gels with single slice, card It is bright its in biosynthetic process not by proteolytic treatment.The weight average molecular weight determined by balancing ultracentrifugation meets glycosyl Change the calculated value of the homodimer of polypeptide.

Embodiment 2

The present embodiment describes to block the anti-MASP-2scFv antibody candidate of the full people of high-affinity of MASP-2 functional activities for identifying Thing is to proceed to the screening technique of affinity maturation.

Background and general principle：

MASP-2 is the compound protein with many individually functional domains, including：One or more MBL and fiber gelatinized egg White binding site, serine protease catalytic site, proteolysis substrate C2 binding sites, proteolysis substrate C4 bound sites Point, the MASP-2 cleavage sites for MASP-2 proenzyme self-activations and two Ca⁺⁺Binding site.ScFv antibody fragments be accredited as with High-affinity combination MASP-2, and identify Fab2 fragments test to determine whether they can block in functional examination MASP-2 functional activities.

In order to block MASP-2 functional activities, antibody or scFv or Fab2 antibody fragments must combine and disturb for The structure epi-position on MASP-2 needed for MASP-2 functional activities.Therefore, the anti-MASP- that many or all high-affinities are combined 2scFv or Fab2 can not suppress MASP-2 functional activities, unless they combine the MASP-2 for being directly related to MASP-2 functional activities On structure epi-position.

The functional examination that the C3 convertase formation of measurement lectin pathway suppresses is used to evaluate " blocking and live for anti-MASP-2scFv Property ".Basic physiological effects of the known MASP-2 in lectin pathway is the next of the complement pathway of generation lectin-mediated Functional component, i.e. lectin pathway C3 convertase.Lectin pathway C3 convertase is crucial multienzyme complex（C4b2a）, its egg Plain boiled water solution cuts C3 into C3a and C3b.MASP-2 is not lectin pathway C3 convertase（C4b2a）Constituent；But, need MASP-2 functional activities are wanted to generate two kinds of protein ingredients（C4b、C2a）, it constitutes lectin pathway C3 convertase.Also, it is right Seem to need above-named MASP-2 all single functional activities to generate lectin pathway C3 convertase in MASP-2. For these reasons, it is believed that the preferred measure for evaluating anti-MASP-2Fab2 and scFv antibody fragments is functional examination, its Measure the suppression of lectin pathway C3 convertase formation.

It is predicted as applying into human body for therapeutic anti-MASP-2 antibody and is obtained after 1mg/kg>90% lectin pathway disappears The target overview removed is the IC in 90% blood plasma₅₀<5nM.In these measure forms in vitro between pharmaceutical active and internal pharmacodynamics Relation carries out experimental verification using anti-rodent MASP-2 antibody.

Select the standard of first generation MASP-2 blocking antibodies for therapeutic use as follows：To MASP-2 high-affinities simultaneously And function IC₅₀Value is up to ~ 25 nM.In addition, screening intersects instead for having with human primate serum and rat blood serum The candidate of answering property.

Method：

For the screening of the scFv phage libraries of MASP-2 antigens.

Antigen：

Used with people MASP-2A histidine-tagged N-terminal 5X and with the histidine-tagged rat MASP-2A of N-terminal 6X Reagent described in embodiment 1 is generated and as discussed previously（Chen etc.,J. Biol. Chem. 276:25894-02 (2001)）Purified by nickel affinity chromatography from culture supernatants.

It is used as the positive control for combining MASP-2 with the anti-MASP-2 antibody OMS100 of the people of Fab2 forms.

Phage library is described：

Light and weight chain variabl area sequence the phage display library of human immunoglobulin(HIg) carries out antigen elutriation（antigen panning）Followed by automatic antibody screening and selection, to identify the height parent for rat MASP-2 albumen and people's MASP-2 albumen With power scFv antibody.

Panning methods：

General introduction：In altogether 3 wheel elutriations, using two kinds of elutriation strategies with the phagocytosis of the separation combination MASP-2 from phage library Body.Two kinds of strategies are related to elutriation in the solution and fish out the bacteriophage with reference to MASP-2.MASP-2 is immobilized on magnetic bead, Via histidine-tagged on target（Use NiNAT globules）Or via the biotin on target（Use Streptavidin globule）.

Preceding two-wheeled elutriation is related to Alkaline Elution（TEA）, and third round elutriation is in conventional alkaline（TEA）Before elution step First with MBL competitiveness elutions.Solid phase is carried out before the 2nd and 3 take turns, and this is to be directed to functional analogue, and classics are mended The C1s and C1r of body approach.After elutriation, the specific enrichment of bacteriophage of the monitoring with the scFv fragments for MASP-2A, and And determine that elutriation strategy has succeeded（Data are not shown）.

ScFv gene clonings from the 3rd wheel elutriation enter pHOG expression vectors, and carry out small-scale filter screening（filter screening）It is as further described below to find the specificity clone for MASP-2A：

Table 7：Bacteriophage Panning methods（Biotin/Streptavidin）

Panning rounds	Antigen (μ g)	Magnetic bead	Block	Pre- elutriation	Elution
						1	Biotin people MASP-2A（10μg）	Streptavidin	4% spot is blocked	Nothing	TEA（Alkalescence）
2	Biotin rat MASP-2A (10 μ g)	Streptavidin	4% spot is blocked	C1s/C1r	TEA（Alkalescence）
						3	Biotin people MASP-2A (1 μ g)	Streptavidin	4% spot is blocked	C1s/C1r	W/MBL is competed（comp）, it is followed by TEA（Alkalescence）

Table 8：Bacteriophage Panning methods（HIS/NiNTA）

Panning rounds	Antigen (μ g)	Magnetic bead	Block	Pre- elutriation	Elution
						1	People MASP-2A adds histidine-tagged (10 μ g)	NiNTA	4% breast in PBS	Nothing	TEA（Alkalescence）
2	Rat MASP-2A adds histidine-tagged (10 μ g)	NiNTA	4% breast in PBS	C1s/C1r	TEA（Alkalescence）
						3	Biotin people MASP-2A (10 μ g)	NiNTA	4% breast in PBS	C1s/C1r	(comp)+TEA is competed with MBL（Alkalescence）

Elutriation reagent：

People MASP-2A

OMS100 antibody（Positive control）

Goat anti human IgG（H+L）（Pierce #31412）

NiNTA globules (Qiagen #LB13267)

Dynabeads M-280 Streptavidins, 10 mg/ml (LB12321)

Normal human serum（LB13294)

The anti-human C3c of multi-clone rabbit（LB13137）

Goat anti-rabbit igg, HRP（American Qualex #A102PU）.

In order to test tagged MASP-2 antigens, tested to capture positive control OMS100 antibody（200ng/ Ml), the MASP-2A or histidine-tagged MASP-2A antigens of the OMS100 antibody respectively with biotin label（10 μg） （NiNTA globules or 200 μ l Streptavidin globules of the 50 μ l in 4% breast PBS）Preincubate.Use Goat anti human IgG（H+L）HRP （1：5000）And TMB（3,3', 5,5'- tetramethyl benzidine）The MASP-2A-OMS100 antibody that substrate detection is combined.

NiNTA globules ELISA is determined

50 μ lNiNTA globules 1ml phosphate buffered salines（PBS）In 4% breast closing and in rotating wheel（rotator wheel）On In incubation at room temperature 1 hour.Abreast, 10 μ g MASP-2A and OMS100 antibody（200ng/ml is diluted in 4% breast-PBS）In advance It is incubated one hour.Then globule is cleaned three times with 1ml PBS-T, and magnet is used between each step.Will be pre- with OMS100 antibody The MASP-2A of incubation is added to the globule of cleaning.Mixture is incubated at room temperature 1 hour in rotating wheel, and magnetic is then used as described above Iron is cleaned 3 times with 1ml PBS-T.Test tube uses 1：5000 are diluted in the Goat anti human IgG of 4% breast in PBS（H+L）HRP is incubated at room temperature 1 hour.For negative control, by Goat anti human IgG in single test tube（H+L）HRP（1：5000）It is added to and cleans and seal The NiNTA globules closed.

Sample is incubated at room temperature 1 hour in rotating wheel, is then cleaned 3 times with 1 ml PBS-T using magnet as described above and is used in combination 1xPBS is cleaned 1 time.Add 100 μ l tmb substrates and in incubation at room temperature 3 minutes.Test tube is placed in 2 minutes small to concentrate on magnetic frame Pearl, is then transferred to microtiter plate by TMB solution and reacts with 100 μ l 2M H₂SO₄Terminate.Read on ELISA readers Take the absorption at 450nm.

Streptavidin globule ELISA is determined

This measure determines progress to the ELISA of NiNTA globules as described above, but uses each μ l Streptavidins of sample 200 small Pearl substitutes and non-biotinylated antigen.

As a result：With positive control OMS100 antibody preincubates plus histidine-tagged and biotin label MASP-2A it is anti- It is each of former successfully to be captured with NiNTA globules or Streptavidin globule respectively.

Elutriation

Three are carried out to scFv phage libraries for histidine-tagged or biotin label MASP-2A as shown in table 7 or table 8 respectively Take turns elutriation.Third round elutriation is eluted with MBL first, then uses TEA（Alkalescence）.In order to monitor the displaying for being directed to target MASP-2A The specific enrichment of the bacteriophage of scFv fragments, proceeds as described below the polyclonal bacteriophage of the MASP-2A for immobilization ELISA。

The MASP-2A ELISA on polyclonal bacteriophage being enriched with after elutriation

The scFv phage libraries for people MASP-2 are carried out after three-wheel elutriation as described above, determined by carrying out ELISA to tool The specific enrichment for having the bacteriophage of the scFv fragments for target MASP-2 is monitored, and the ELISA is by for fixation Carried out in the polyclonal bacteriophage colony of the enrichment of the MASP-2 elutriations generation of change, as described below.

Method：

5ng/ml MASP-2A are immobilized on the maxisorpELISA plates in PBS at 4 DEG C overnight.From all three-wheel elutriations Packaging bacteriophage 1：3 are diluted in 4% breast-PBS, and are titrated with 3 times of dilutions.Negative control is M13 helper phages.

Closing is 4% breast in PBS.Between each step, plate is in 200 μ l PBS- tweens 0.05%（v/v）It is middle to clean 3 times. Primary antibody is rabbit α-fd（M13 coat protein）, 1：5000 in 4% breast-PBS（w/v）In.Conjugate is rabbit α-goat-HRP with 1： 10000 in 4% breast-PBS（w/v）In.Substrate is ABTS.All volumes, are 100 μ l/ holes except cleaning and closing.It is all to be incubated It is to be shaken 1 hour in room temperature.

As a result：

The result of Phage-ELISA shows the specific enrichment for MASP-2A scFv for two kinds of elutriation strategies.Referring to figure 2.Captured as shown in Fig. 2 strategy is related to by NiNTA magnetic beads, the phagocytosis for MASP-2 is obtained after two-wheeled elutriation ScFv enrichments on body, and it is respectively provided with good enrichment in competitive and TEA elutions in third round elutriation latter two strategy.It is cloudy Property control bacteriophage be M13 helper phages, its show under its minimum dilution is directed to MASP-2A no cross reactions.These knots Signal observed by fruit proves is due to scFv specific bindings MASP-2.

Filter is screened：

The bacterial clone of the plasmid comprising coding scFv fragments from third round elutriation is selected, nitrocellulose filter is gridded to Above and on non-induced culture medium grow overnight to produce main flat board（master plate）.Selected altogether from third round elutriation 18000 are cloned and analyzed, and half comes from competitive elution and half comes from subsequent TEA elutions.

Nitrocellulose filter with bacterial clone is induced to express and secrete solvable scFv albumen with IPTG, and makes it 4% breast in contact the second nitrocellulose filter of MASP-2A antigen coats, the also useful PBS contacted together（Confining liquid）Coating Parallel progress film.

Examined with reference to MASP-2A scFv via their c-Myc labels with mouse α-cMyc mAb and rabbit α-mouse HRP Survey.Selection is to tackling hitting for further expression and subsequently for positive and to breast-PBS feminine genders the scFv clones of MASP-2A Elisa assay.

As a result：For elutriation then scFv conversions and the filter screening acquisition 137 of MASP-2A scFv phagemid libraries Positive colony.Using both NiNTA and Streptavidin strategy, most of positive colony comes from the competitive elution with MBL. All positive colonies proceed micro- expression（200 μ l scales）And subsequent extracted.By using sucrose lysis buffer and lysozyme It is incubated bacterial suspension（Supernatant is separated by centrifugation step afterwards）ScFv is separated from bacteria periplasm.Comprising with pericentral siphon The tolerant scFv being secreted into together in culture medium supernatant is determined by two kinds and analyzed：Use the MASP-2A of physical absorption Binding analysis of ELISA and the MASP-2A of use amine coupling the CM5 chips on Biocore, as described in more detail below.

MASP-2A ELISA in the scFv candidate clones by elutriation/scFv conversions and filter Screening and Identification

Method：

4 μ g/ml MASP-2A are immobilized in the maxisorpELISA plates in PBS（Nunc）On 4 DEG C overnight.Second day, plate led to Cross and use PBS- tweens（0.05%）Clean 3 times and closed.From 137 scFv candidates（It is generated as described above）Each thick ScFv materials（100 μ l culture mediums-periplasmic extract）It is added to each hole of plate.Next, adding anti-cMyc and finally walking The conjugated rabbit anti-mouse antibody of HRP is applied to detect the scFv of combination in rapid.Reaction walks ABTS in peroxidase substrate 1- （Calbiochem）Middle colour developing.Positive control is the OMS100 that 10 μ g/ml are diluted in PBS- tweens 0.05%（With Fab2 lattice The anti-MASP-2 antibody of formula）.Negative control is culture medium-pericentral siphon that plasmid is free of from XL1- indigo plants.

Cleaned between each step with 3x200 μ l PBS- tweens 0.05% (v/v).

Primary antibody is mouse α-cMyc, 1:5000 in PBS- tweens 0.05% (w/v).

Conjugate is with 1:5000 Goat anti human IgG (H+L, the Pierce in PBS- tweens 0.05% (w/v) 31412).Substrate was ABTS, in incubation at room temperature 15 minutes.All volumes, are 100 μ l/ holes except cleaning and closing.It is all to incubate Educate is shaken 1 hour in room temperature.

As a result：108/137 clone is positive in this ELISA measure（Data are not shown）, wherein 45 clones are as follows The further analysis.Positive control is the OMS100 Fab2 that 10 μ g/ml are diluted in PBS- tweens, and this clone is Positive.Negative control is culture medium-pericentral siphon that plasmid is free of from XL1- indigo plants, and it is negative.

Embodiment 3

This embodiment describes to block in normal human serum for analyzing the anti-MASP-2 scFv antibody candidates of the full people of high-affinity The MASP-2 functional screening methods of MASP-2 activities.

General principle/background

Measure the measure of the suppression of lectin pathway C3 convertase formation

The functional examination that the C3 convertase formation of measurement lectin pathway suppresses is used to evaluate anti-MASP-2 scFv candidate clones " blocking activity ".Lectin pathway C3 convertase is multienzyme complex（C4b2a）, it is potent proinflammatory into two that its proteolysis cuts C3 The C3b of property fragment, anaphylatoxin C3a and hair conditioning.In terms of transmitting inflammation, the formation of C3 convertase seemingly lectin pathway Committed step.MASP-2 is not lectin pathway C3 convertase（C4b2a）Constituent；Therefore anti-MASP-2 antibody（Or Fab2）The activity of C3 convertase existed will not directly be suppressed.But need MASP-2 serine proteases with Generate two kinds of protein ingredients（C4b, C2a）, it constitutes lectin pathway C3 convertase.Therefore, MASP-2 functional activities are suppressed Anti- MASP-2 scFv（That is the anti-MASP-2 scFv of blocking property）The from the beginning formation of lectin pathway C3 convertase will be suppressed.C3 bags Containing part of the uncommon and highly reactive thioester substrate as its structure.C3 is cut by C3 convertase in this measure Afterwards, the thioester substrate on C3b can be with the hydroxyl or amino that are immobilized in the macromolecular of plastic eyelet bottom via ester or amido link Covalent bond is formed, so as to facilitate detections of the C3b in ELISA measure.

Yeast mannans are the activators of known lectin pathway.In the method for following measurement C3 convertase formation In, the plastic eyelet of coating mannosan is incubated to activate lectin pathway together with the human serum of dilution.Then clean-out opening and make The C3b being immobilized on hole is determined with standard ELISA assay.This C3b generated in determining amount directly reflects agglutinin way The from the beginning formation of footpath C3 convertase.Tested in measure selected concentration anti-MASP-2scFv suppress C3 convertase formation and with The ability that C3b is generated afterwards.

Method：

45 candidate clones identified as described in example 2 above are expressed, purified and are diluted to identical storage concentration, and it is dilute again Release comprising Ca⁺⁺And Mg⁺⁺GVB buffer solutions（4.0 mM barbitals, 141 mM NaCl, 1.0 mM MgCl₂, 2.0 mM CaCl₂, 0.1% gelatin, pH 7.4）In with ensure it is all clone have same amount of buffer solution.ScFv clones are each with 2 μ g/ Ml concentration is tested in triplicate.Positive control is OMS100Fab2 and tested with 0.4 μ g/ml.In presence and not C3c is monitored in the case of there is scFv/IgG clones to be formed.

Mannosan is in 50mM carbonate buffer solutions, pH9.5 (15mM Na₂CO₃ + 35mM NaHCO₃ + 1.5 mM NaN₃) in be diluted to the concentration in 20 μ g/ml (1 μ g/ holes), and be coated on elisa plate 4 DEG C overnight.Next day, sweet dew gathers The plate of glycolyx 200 μ l PBSs 3 times.Then 100 μ l 1%HSA confining liquids are added in hole and small in incubation at room temperature 1 When.Plate is stored on ice until addition sample with 200 μ l PBSs 3 times and with 200 μ l PBS.

Normal human serum is diluted to 0.5% in CaMgGVB buffer solutions, and scFv clones or the OMS100 Fab2 positives are right It is added in triplicate in this buffer solution according to 0.01 μ g/ml, 1 μ g/ml (only OMS100 controls) and 10 μ g/ml, and It is added to before the elisa plate of closing in preincubate 45 minutes on ice.By being incubated initiation reactions in 1 hour and by inciting somebody to action at 37 DEG C Plate is transferred to ice bath termination.With the subsequent goat α of rabbit α-mouse C3c antibody-rabbit HRP detection C3b depositions.Negative control is free from resisting Body（Without OMS100=maximum C3b depositions）Buffer solution, and positive control is with EDTA（Without C3b depositions）Buffer solution. Background is determined by carrying out identical measure, but in the negative hole of mannosan.For the back of the body of the plate without mannosan Scape signal is subtracted from mannosan positive signal.Cut-off standard is arranged on uncorrelated scFv clones（VZV）Only buffer solution The half of activity.

As a result：Based on cut-off standard, such as Fig. 3 A and 3B are shown, find the activity of 13 clone's blocking MASP-2 altogether.Choosing Select all generations>13 of 50% approach suppression clone and are sequenced, and obtain 10 unique clones, as shown in Table 9 below.It was found that 10 different being cloned in during complement is determined shown in table 9 cause acceptable functional activity.It was found that all 10 clones tools There is an identical light chain subclass, λ 3, but three different heavy chain subclasses, VH2, VH3 and VH6.The Germline sequences of these clones Sequence identity is also shown in table 9.

Table 9：10 unique clones with the anti-MASP-2 activity of feature

As shown in table 9 above, there is acceptable functional activity clone different with 10 of unique sequences to carry out for selection Further analysis.As being previously mentioned in table 9, based on identical sequence, some clones are detected two or three times（Have referring to table 9 There is the first row of clone name）.

The expression and purifying of ten svFv candidate clones

Ten candidate clones shown in table 9 in nickel dam analysis are purified with one liter of scale expression and via ion exchange.At that Afterwards, the sample each cloned runs to evaluate monomer and dimer content on size exclusion chromatography post.Such as institute in table 10 below Showing, nearly all scFv clones exist with monomeric form, and separate this monomer fraction is used to further test and grade.

Table 10：Content of monomer is analyzed

The combination of test unit fraction and functional activity

The clone shown in table 10 is expressed with 1L scales, is purified on metal chromatography and ion exchange, is passed through size exclusion chromatography （SEC）Separating monomer fraction, and repeat function determined to determine IC₅₀Value and cross reactivity.

The functional examination of monomer fraction

The monomer fraction and the test function IC in being serially diluted of preceding ten clones shown in purifying table 10₅₀NM, wherein often The individual GVB buffer solutions and human serum for cloning the calcic and magnesium that receive same concentrations.ScFv is cloned in triplicate in 12 dilutions Tested.Positive colony is OMS100 Fab2.C3b depositions are monitored in the case of presence or absence of antibody.As a result it is shown in In table 11 below.

Combination mensuration:

The binding affinity K of the monomer fraction of the purifying of ten candidate scFv clones is determined with two kinds of distinct methods_D。MASP-2A Either be immobilized on CM5 chips by amine coupling or solid concentration scFv（50nM）The high affine of amine coupling is used first Power α-cMyc antibody captures, and the concentration series of the MASP-2A in solution is passed through chip.As a result it is shown in table 11 below In.

As a result：

Table 11：For the function inhibitio activity of the ten candidate scFv clones determined with free state（IC₅₀）Combined with MASP-2 Affinity（K_D）Summary

Discussion of results

As shown in figure 11, in functional examination, 5 in 10 candidate scFv are given less than 25nM targets using 0.5% human serum The IC of standard₅₀NM values.As described below, these are cloned in there is human primate serum and rat blood serum in the case of Further test to determine the functional activity in other species.On binding affinity, all binding affinities in the solution All in low nM scope or more preferable, and use in the MASP-2 of immobilization conventional determining, only two clones（4D9 and 17D20） There is affinity in low nM scopes.The observation result of more high-affinity is probably to resist in the solution in the measure based on solution What the fact that former multimerization, caused.Also, when target is immobilized on chip（Via orientation coupling）When, epitope may be shielded, So as to reduce the affinity observed in immobilization measure.

Embodiment 4

The anti-MASP-2scFv clones of this embodiment description ten candidates of test and the result of rat MASP-2 cross reactivity With the IC that these scFv clones are determined in functional examination₅₀The result of value, the functional examination determines them in human serum, inhuman Suppress the ability for the complement activation that MASP-2 is relied in primate serum and rat blood serum.

Method：

With rat MASP-2 cross reactivity

Ten times shown in the table 9 for testing embodiment 3 in being determined for the rat MASP-2A of absorption conventional ELISA ScFv clones are selected for rat MASP-2A cross reactivity.Rat MASP-2A is diluted to 4 μ g/ml and is coated in PBS Maxisorp elisa plates（Nunc）On 4 DEG C overnight.Second day, plate passed through in PBS- tweens（0.05%）Middle 3 progress of cleaning Closing.20 μ g/ml scFv clones are diluted in PBS- tweens（100μl）It is added to plate, and further with 4 times of dilution drops It is fixed three times.With anti-cMyc and rabbit anti-mouse HRP secondary antibodies detection MASP-2A specificity scFv clones（ScFv comprising combination Hole）.Reaction is in peroxidase substrate TMB（Pierce）Middle colour developing.Positive control is that 10 μ g/ are diluted in PBS- tweens Ml OMS100 Fab2.The clone's display and rat MASP-2A cross reaction of all tests, it is it is contemplated that because second Take turns elutriation rat MASP-2（Data are not shown）.

Ten candidate scFv are cloned in human serum, non-human primate（NHP）Menu in serum and rat blood serum Levy

The determination of baseline C3c levels in different serum

First, experiment is carried out as follows to compare in three kinds of serum（People, rat and NHP）Middle baseline C3b levels.

Mannosan is diluted to 20 μ g/ml and is coated on elisa plate at 4 DEG C overnight.Hole is closed with 1%HSA within second day. Normal person, rat and cercopithecus aethiops serum（Non-human primate " NHP "）Serum is diluted to since 2% with 2 times of dilution methods In CaMgGVB buffer solutions.By being incubated initiation reaction in 1 hour at 37 DEG C and being terminated by the way that plate is transferred into ice bath.It is small with rabbit-anti The subsequent goat antirabbit HRP detections C3b depositions of mouse C3c antibody.Negative control is free from antibody（Maximum C3b is caused to sink without OMS100 Product）Buffer solution, and suppress positive control be with EDTA（Without C3b depositions）Buffer solution.

Three kinds of serum of Fig. 4 illustrations（People, rat and NHP）The baseline of middle C3c levels.As shown in figure 4, being tested Different serum in C3c levels it is very different.When comparing C3c levels, it appears that 1% human serum is provided such as 0.2%NHP and 0.375% The equivalent level of rat blood serum.Based on these results, the concentration of serum be normalized allow scFv results at three kinds not Directly compare in the serum of same type.

The functional examination of scFv clones in different serum

Then the monomer fraction of purifying of ten candidate scFv clones is tested in human serum, rat blood serum and cercopithecus aethiops serum （Non-human primate " NHP "）Middle function IC₅₀nM.It is measured, is purified using 1000nMscFv as described in example 3 above Albumen and it is any be diluted in CaMgGVB buffer solutions 0.9% normal human serum, or in CaMgGVB buffer solutions dilute To 0.2% cercopithecus aethiops serum, or it is diluted in CaMgGVB buffer solutions 0.375% rat blood serum.All ten scFv grams Grand to be tested in dilution series, wherein they receive the GVB buffer solutions and serum containing calcium and magnesium of same concentrations.scFv It is cloned in 12 dilutions and is tested in triplicate.Positive control is added in 100ng/ml OMS100 Fab2 or to reaction Plus EDTA.Negative control is incoherent scFv controls or the PBS without scFv.Presence or absence of scFv or Fab2 antibody In the case of monitor C3b deposition.Subtracted in 100ng/ml OMS100 background signal from all signals.Table 12 is summarized The result of functional examination in all three serum.

Table：12：The function IC that scFv is cloned in three kinds of different types of serum₅₀(nM) it is active.

Note：* in human serum（Test #1）On first group of data completed on unconcentrated scFv samples, therefore, with low The clone of concentration can not fully titrate.In remaining experiment, all clones are concentrated and titrated to be started in same concentrations.

The result of the functional activity of scFv candidate clones is summarized in different serum：

After serum on C3b deposition levels to be normalized, in people and non-human primate（NHP）Own in serum Ten scFv clone equal display function.Six in human serum most active clones are：When from preferably to during worst arrangement, 9P13>17N16>17D20>4D9>3F22>18L16.In NHP serum, clone is arranged as（Preferably arrive worst）：17L20> 17N16>17D20>9P13>18L16>3F22.17N16 and 17D20 comes first three in people and NHP serum.17D20 is also big Some activity are shown in mouse serum.

Based on these results, first three scFv clones are defined as：18L16,17D20 and 17N16.As shown in table 13 below, this Three clones are further in the human serum of dilution（1% serum）Middle analysis.

Table 13：The C3 of three candidate clones is determined：Dilute serum（1%）In (IC₅₀ nM)

Fig. 5 A are the amino acid sequences that total length scFv clones 17D20,18L16,4D9,17L20,17N16,3F22 and 9P13 Compare.ScFv clones include weight chain variable district（Amino acid/11-120）, connector area（Amino acid/11 21-145）And light chain variable district（Ammonia Base acid 146-250）.As shown in Figure 5A, the heavy chain region of most active clone（Residue 1-120）Amino acid alignment disclose Two different components do not belong to VH2 and VH6 gene families.As shown in Figure 5A, the clone on VH2 classes：17D20,18L16 and 4D9 VH areas 20 amino acid positions in 120 amino acid areas altogether have variation（That is 83% homogeneity）.

As further shown in figure 5, the clone on VH6 classes：17L20,17N16,3F22 and 9P13 VH areas are altogether 18 amino acid positions have variation in 120 amino acid areas（That is 85% homogeneity）.

Fig. 5 B are the sequence alignments that scFv clones 17D20,17N16,18L16 and 4D9.

Table 14：The sequence of the scFv candidate clones shown in Fig. 5 A and 5B

Ordering is（1）Human blood cleaning function potency and full blocking；（2）NHP cross reactivities and（3）Sequence polymorphism. Selection 17D20 and 17N16 is used as the optimal representative from each gene family.18L16 is selected as with appreciable CDR3 3rd candidate of sequence polymorphism.

Due to complete function blocking, 17N16 and 17D20 are the first two selections, are imitated with the best function for people Valency；Appreciable monkey cross reactivity and different VH gene families.Because VH sequences are almost identical with 17N16, so removing 3F22 and 9P13.Due to gentle potency, so removing 18P15,4J9 and 21B17.17L20 does not continue, because its only part Block.

There is similar active and appreciable diversity compared to 17D20,18L16 and 4D9.18L16 is selected because than 4D9 Higher primate cross reactivity.

Therefore, based on these standards：Following three mother clone：17D20,17N16 and 18L16 enter further described below Affinity maturation.

Embodiment 5

This embodiment describes three female clone 17D20,17N16 and 18L16（Identified as described in embodiment 2-4）Clone into open country Raw type IgG4 forms and the female features cloned as total length IgG of evaluation three.

General principle：

Identify that the anti-MASP-2scFv of total length people with functional medium potency resists using phage display as described in embodiment 2-4 Body.These three mother clone 17D20,17N16 and 18L16 are selected to be used for affinity maturation.In order to evaluate these mother clones as complete Long IgG feature, generates the IgG4 wild types and S228P hinge area IgG4 mutant forms of these antibody.Include S228P Hinge region mutations body is to increase serum stability（Referring to Labrijn A.F. etc.,Nature Biotechnology 27:767 (2009)）.

The amino acid sequence of IgG4 wild types is described as SEQ ID NO:63, by SEQ ID NO:62 codings.

IgG4 S228P amino acid sequence is described as SEQ ID NO:65, by SEQ ID NO:64 codings.

IgG4 molecules are also cut into the forms of F (ab') 2 with pepsin digestion, and by size exclusion chromatography be classified with Female clone is set directly to be compared with OMS100 control antibodies, the latter is F（ab）2 molecules.

Method：

Clone is set to generate total length form

Three female Cloning Transformations are wild type IgG4 forms and IgG4 mutant S228P forms.This is achieved by the following procedure：Will be appropriate VH and VL regions separated from above-cited female clone PCR and they be cloned into containing appropriate heavy chain constant region PcDNA3 expression vectors are to create fused in-frame to produce desired antibody.It is right with three that are mutated IgG4 forms female clones Afterwards with pepsin cutting to produce the fragments of F (ab') 2, and the latter in size exclusion chromatography column fractionation by being purified.

Combination mensuration

The candidate mother clone for changing into IgG4 forms is transiently transfected into HEK293 cells and the supernatant from transient transfection exists ELISA is titrated in determining.Clone's display reactivity outstanding with the people MASP-2A of physical absorption is simultaneously arranged in the following order： 17N16>17D20>18L16 (data are not shown).

Then purified colonies and the re-test in following ELISA and determination of activity.People MASP-2A is with 3 μ g/ml in PBS In be coated on maxisorp plates, IgG（45g/ml）It is diluted to 300nM's in PBS- tweens with Fab'2 (30 μ g/ml) Start concentration and further 3 times of dilutions.Goat α-the human IgG being conjugated with HRP（Southern Biotech）IgG is detected, and Goat α-human IgG the H+L being conjugated with HRP（Pierce 31412）Detect F (ab') 2.Reaction is developed the color with tmb substrate to be used in combination 2MH₂SO₄Terminate.As a result it is shown in table 15 below.

Table 15：To people MASP-2 binding affinity

Functional examination

Using as described in example 4 above use 1% normal human serum（NHS）C3 convertase determine to compare female scFv grams of clone The functional activity of total length IgG4 homologues grand and in 1%NHS.Mannosan is diluted to 20 μ g/ml concentration and is coated on On elisa plate at 4 DEG C overnight.Hole is closed with 1% human serum within second day.Human serum is in CaMgGVB buffer solutions and the antibody of purifying It is diluted to 1%, scFv (900 nM), F (ab') 2 (300 nM), IgG (300 nM) duplicate with different dilution factors series It is added to same amount of buffer solution, and in preincubate 45 minutes on ice before the elisa plate of closing is added to.By 37 DEG C it is incubated and initiation reaction in 1 hour and is terminated on ice by the way that plate is placed in.It is true with the subsequent goat α-rabbit HRP of rabbit α-mouse C3c antibody Determine C3b depositions.The background of OMS100 in mannosan positive plates with 50nM is subtracted from curve.This analysis result it is total Knot is shown in table 16 below.

Table 16：Determined using the C3 convertase of 1% human serum（IC₅₀nM）

Note：Two values shown in the 2-4 row of table 16 refer to two results individually tested.

To each clone, the function potency of IgG4 mother clones also with IgG4 hinge mutation bodies（S228P）Compare.Use 1% The digital IC that NHS C3b depositions are determined₅₀Value is shown in table 17 below.

Table 17：In 1% human serum（IC₅₀）In C3b deposition in wild type（IgG4）Opposite from hinge mutant form （S228P）

As described in upper table 17, in some cases, inexpectancy is noticed for the IgG' of the scFv derived from Antagonism Activator pharmacology.Mechanism to this observation result not yet understands.

There is the activity of female clone for the IgG4 conversions for suppressing function in 1%NHS in more rigorous condition determination（More connect Near-lying mode intends physiological condition）Under carried out further evaluation.In order to assess antibody activity in physiological conditions, in rigorous measure Under the conditions of, use minimum dilution（90%）Human plasma, tests it to mother's clone's IgG4 prepared products and suppresses lectin pathway（LP）According to Bad C3b deposits to the ability of the coated plate of mannosan.

The result that C3b depositions in 90% human plasma are determined is shown in figure 6.Due to MASP-2 and its substrate with than Diluting plasma is present in measure mixture in determining using high about 100 times in 1% normal human serum of concentration, is generally expected to short of money Anti-agent dosage-response curve is moved to right.As shown in Figure 6, OMS100 and the MASP-2 antibody of all tests are observed as expected Moving to right to lower apparent potency.Surprisingly however it was, for 17D20 hinge region mutations body（S228P）Do not observe To the reduction of apparent potency, and potency in this form is measured suitable with 1% blood plasma（Referring to table 17）.90% NHS is determined in form, finds 17D20 IgG4（S228）Function potency ratio OMS100Fab2 it is lower slightly, this with 1%NHS Measurement result is on the contrary, wherein OMS100 is higher 50 to 100 times than 17D20IgG4 S228P effect（Data are not shown）.17N16 open country Raw type IgG4 forms display that complete suppression in 90%NHS, but it is relatively low that somewhat potency in form is determined at this（IC₅₀ ≈ 15nM）, and 18L16 wild type IgG4 form potency is relatively low and only part suppresses, as shown in Figure 6.

Based on these discoveries, the activity of female clone of IgG4 conversions is by under rigorous condition determination（90%NHS）Check C4b depositions are further to be evaluated.This determines form on providing direct survey by the antibody activity of the MASP-2 enzyme reactions being catalyzed Amount.

Measure the measure of the suppression for the C4 cuttings that MASP-2 is relied on

Background：MASP-2 serine protease is high degree of specificity, and has identified only two MASP-2 egg White background thing, C2 and C4.C4 cutting generation C4a and C4b.Anti- MASP-2 Fab2 can combine the structure epi-position on MASP-2, should Epitope is directly related to C4 cuttings（For example, binding sites of the MASP-2 to C4；MASP-2 serine protease catalytics site）And by This suppresses MASP-2 C4 cutting functions activity.

Yeast mannans are the activators of known lectin pathway.In following measurement MASP-2 C4 cleavage activities In the method for formation, the plastic eyelet of coating mannosan is incubated 90 minutes to activate agglutinin at 4 DEG C together with 90% human serum Approach.Then clean-out opening and determined using standard ELISA assay and be immobilized in people C4b on hole.This C4b's generated in determining Amount is measuring for the C4 cleavage activities that MASP-2 is relied on.This is tested in determining in their suppressions of the anti-MASP-2 antibody of selected concentration The ability of C4 cuttings processed.

Method：Adhesion plate is with being diluted in 1.0 μ g/50 μ in 50mM carbonate buffer solutions, pH9.5 in 96 hole Costar The mannosan in L/ holes is in 5 DEG C of overnight incubations.Per hole with 200 μ L PBSs 3 times.In the PBS of hole then with 100 μ L/ holes 1% bovine serum albumin(BSA) is closed and gently mixes incubation 1 hour in room temperature.Per hole with 200 μ L PBSs 3 times.Anti- MASP-2 resists Body sample is containing Ca⁺⁺And Mg⁺⁺GVB buffer solutions（4.0 mM barbital、141 mM NaCl、1.0 mM MgCl₂、2.0 mM CaCl₂, 0.1% gelatin, pH 7.4）In the concentration of selection is diluted at 5 DEG C.90% human serum is added to above-mentioned sample at 5 DEG C In, and shift 100 μ L to every hole.Plate covers and is incubated 90 minutes in ice-water bath to allow complement activation.By to reaction Mixture adds EDTA terminating reactions.Per hole PBS polysorbas20s（0.05% polysorbas20 in PBS）The μ L of 5 x 200 are cleaned, then Cleaned 2 times with 200 μ LPBS per hole.100 μ L/ holes biotin-conjugateds the anti-human C4c of chicken (Immunsystem AB, Uppsala, Sweden) 1：700 dilutions are added to comprising 2.0mg/ml bovine serum albumin(BSA)s（BSA）PBS in and it is gentle mixed at room temperature Close and be incubated 1 hour.Per the hole μ L of 5 x of PBS 200.The strepto- parent of the μ g/ml of 100 μ L/ holes 0.1 peroxidase conjugated It is added to plain (Pierce Chemical #21126) in the PBS comprising 2.0mg/mlBSA and warm on shaking table at room temperature It is incubated 1 hour with mixing.Per the hole μ L of 5 x of PBS 200.Add the peroxidase substrate TMB in 100 μ L/ holes （Kirkegaard ＆ Perry Laboratories) and in incubation at room temperature 16 minutes.Peroxidase reaction passes through addition 100 The 1.0 M H in μ L/ holes₃PO₄Terminate and measure OD₄₅₀。

As a result：

In this form, 17,D20 two kinds of IgG4 forms suppress the C4b depositions of lectin pathway driving, although IC₅₀Value ratio C3b depositions determine high about 3 times.It is interesting that 17N16 IgG4 wild types show good activity in this measure, have and C3b Deposition determines suitable IC₅₀Value and dosage-response overview.The obvious potency of 18L16 is lower and has been not carried out in this form Full suppression（Data are not shown）.

Discuss：

As described in embodiment 2-5, the anti-MASP-2scFv antibody of full people with function blocking activity is entered using phage display Row identification.These three are selected to clone, 17N16,17D20 and 18L16 are used for affinity maturation and further test.In order to evaluate These mothers clone the feature as total length IgG, and the IgG4 wild types and IgG4 S228P hinge areas for generating these antibody are dashed forward Deformation type.Described in such embodiment, when determined with the standard feature of 1% human plasma tested in form when, most of total length IgG has improved functional activity compared with their scFv homologues.In order to estimate the antibody activity under physiological condition, make With the test that female clone's IgG4 prepared products are carried out under the rigorous condition determination of 90% human plasma.Under these conditions, based in standard （1%）Their performance in blood plasma functional examination, several antibody, which are disclosed, is substantially better than expected function potency.

Embodiment 6

Reset and affinity maturation this embodiment describes female clone 17D20,17N16 and 18L16 chain, and the son obtained The analysis of clone.

Method：

In order to identify the antibody with improved potency, three mother scFv being identified as described in embodiment 2-5 clone 17D20, 17N16 and 18L16 carries out light chain rearrangement.This process is related to matches the children from six healthy donors by the VH of each female clone It is young, mankind's lambda light chain（VL）Library composition combinatorial libraries generation.Then screening this library has improved binding affinity And/or functional scFv clones.

Each mother's clonal analysis clone of 9000 light chain rearrangements, altogether 27000 clones.Induction is cloned per height Express and secrete solvable scFv and screen the ability with reference to people MASP-2A.With reference to people MASP-2A scFv via their c- Myc labels are detected.This initial screening obtains the selection of 119 clones altogether, and it includes 107 sons gram from 17N16 libraries It is grand, 8 son clones from 17D20 libraries and 4 son clones from 18L16 libraries.

119 clone to express on a small scale, on NiNTA posts purifying and ELISA measure in testing needle to physical absorption People MASP-2A binding affinity.

As a result：

ELISA measurement results on the representative subset of 119 son clones are shown in Fig. 7 A and B.Fig. 7 A illustrations On the 17N16 mothers clone result that son clone determines to the ELISA that people MASP-2A is titrated relatively.Fig. 7 B illustrations on The 17D20 mothers clone result that son clone determines to the ELISA that people MASP-2A is titrated relatively.

As shown in Figure 7 A, the son clone 17N16m_d16E12 and 17N16m_d17N9 tools derived from 17N16 mother clones There is the affinity higher than female clone.Also, as shown in Figure 7 B, a clone 17D20m_d18M24 derived from 17D20 mother clones With the affinity higher than female clone.These three clones and three extra clones：17N16m_ with low expression level D13L12,17N16m_d16K5,17N16m_d1G5 and 17D20m_d1824 are expressed with 0.5L scales, pass through size exclusion chromatography Purify into monomer fraction and the re-test in ELISA and functional examination.18L16 libraries do not produce any with desired knot The son clone for closing affinity.

After purification, the inhibitory activity that 6 sons of test are cloned in complement measure.As a result it is shown in Table 18.

Table 18：The complement of mother clone and son clone are determined

As shown in upper table 18, only one 17N16m_d17N9 has such as the affinity of female clone's same range in these clones And activity.

Fig. 8 is total length scFv mother clone 17N16 (SEQ ID NO:59) with 17N16m_d17N9 clones (SEQ ID NO:66) amino acid alignment, shows light chain between the two clones（Since SYE）With 17 amino acid residues not Together.

Several extra candidate son clones are obtained to screening again for 17N16 λ libraries, wherein being determined in ELISA and complement In identify 17N16m_d27E13, and be included in the set of candidate son clone and be used to further analyze.

Son clone is determined in different serum

Candidate son clone is analyzed in different serum as follows.Mannosan is diluted to 20 μ g/ml and is coated on elisa plate 4 DEG C overnight.Hole is closed with 1%HSA within second day.Cercopithecus aethiops serum-dilution is to 0.2% in CaMgGVB buffer solutions, and rat serum is thin Release to 0.375% and human serum is diluted to 1%.The scFv of the purifying of each from candidate son clone is duplicate with difference Concentration series are added in same amount of buffer solution, and in preincubate 45 minutes on ice before the elisa plate of closing is added to. By being incubated initiation reaction in 1 hour at 37 DEG C and being terminated by the way that plate is transferred into ice bath.With rabbit α-subsequent goat of mouse C3c antibody α-rabbit HRP detection C3c releases.Subtracted in mannosan female plate with 0.1 μ g/ml OMS100 background from these curves Go.As a result it is summarised in table 19 below.

Table 19：The IC of mother clone 17N16 and son clone 17N16m_d17N9 and 17N16m_d27E13 in different serum₅₀ Value

Note：Two values shown in the 2-4 row of table 19 refer to two results individually tested.

Discussion of results:

As shown in table 19, sub- clone 17N16m_d17N9 has the functional activity higher than female clone.Except compared with mother clone In light chain outside the difference of 17 amino acid sequences, the function of improving in rat blood serum is so that this clone is positive candidate.Base In this data, selection son clone 17N16m_d17N9 is used to further analyze.

Embodiment 7

Son clone 17D20m_d3521N11 of this embodiment description derived from female clone 17D20 generation and analysis.

Background/general principle：

In order to improve female clone candidate 17D20mc affinity, carried out in the CDR3 of heavy chain first three amino acid extra " look-through mutagenesis ".This is the mutation activity parallel with 17D20mc normal light chain rearrangement.Therefore, three are built not by PCR With scFv libraries, wherein using degenerate codon by the set of amino acid position 1,2 and 3 pairs of all possible 20 kinds of amino acid Carry out randomization.After clone library, carry out micro- scale expression and monitoring scFv is combined on coating MASP-2A CM5 chips （Do not show）.To carrying out micro- scale in the library different with three of 3 randomizations of position 1,2 on coating MASP-2A chip The BIAcore analyses of expression, and identify potential interesting son clone.

It was observed that for CDR-H3 amino acid position 1,2 and 3, not finding clone compared to female candidate clone 17D20m tools There is improved dissociation yield.But in CDR-H3 amino acid position 3 there are some candidates of mutation to illustrate compared to female candidate gram The dissociation yield that grand 17D20m improves.These clones（#35, #59 and #90）It is sequenced to identify mutation.Two derived from " browsing prominent The sequence and 17D20mc of the clone of change "（Original series）Compare.Ironically, the clone of all sequencings is except one（#90） Replace compared to female candidate comprising Ala-Arg.

Fig. 9 is amino acid sequence (the SEQ ID NO of scFv mother clones 17D20m heavy chain region:18 amino acid 61-119） With having the scFv of mutation to clone in CDR-H3：Clone #35（SEQ ID NO:20 amino acid 61-119, in SEQ ID NO: 18 position 102 has substitutions of the R to A）, clone #59（With clone's #35 identical sequences）And clone #90（SEQ ID NO: 18 position 102 has substitutions of the P to A）The sequence of amino acid sequence in CDR-H3 areas compare.

Mutant clone #35 and #59 analysis

Mutant clone #35 and #59 expresses pacing examination of going forward side by side with small-scale, in immobilization MASP-2A（10 μg/ml）Drop Determine in ELISA to be compared with female candidate clone 17D20.ScFv is serially diluted and used anti-Myc for 5 times since 20 μ g/ml（It is small Mouse）/ anti-mouse HRP is detected.Mother candidate clone 17D20 is compared with #59 for candidate clone #35 to see in ELISA measure Observe the combination slightly improved（Data are not shown）.

Improved clone #35 is combined with the clone 17D20m_d21N11 of optimal light chain rearrangement.Candidate 17D20md35 (Ala-Arg) the mutation in VH is combined with candidate 17D20m_d21N11 light chain, so as to obtain referred to as VH35-VL21N11's Clone, otherwise referred to as 3521N11.

Figure 10 A are the sequences in female clone 17D20 CDR3 areas（SEQ ID NO:18 amino acid 61-119）, with identical The mutant clon #35 of son clone's 17D20m_d21N11 same areas of sequence and combination 17D20m_d21N11 VL（Referred to as “3521N11”）Same area (SEQ ID NO:20 amino acid 61-119) amino acid alignment.Highlighted VH sequences Region includes CDRH3, and the target full region being mutated is underlined.

Figure 10 B are to include total length scFv (the SEQ ID NO in the VL and VH areas of 17D20 mother clones:55) cloned with son 17D20m_d21N11 (SEQ ID NO:67) protein sequence is compared.ScFv clones 17D20m_d3521N11 is described as SEQ ID NO:68.Note：It is " E " to have determined the X residues in Figure 10 B in position 220, such as SEQ ID NO:Described in 68.

The titration ELISA of the scFv shown in Figure 10 set is determined in MASP-2（10 μg/ml）Upper operation.As a result show In table 20.

Table 20：ELISA on people MASP-2

The functional activity that further analysis 17D20m_d3521N11 are cloned described in following article embodiment 8.

Embodiment 8

This embodiment describes candidate clone 17N16m_d17N9 and 17D20m_d3521N11 to IgG4, IgG4/S228P and IgG2 The conversion and analysis of form.

General principle/background

Antibody screening method described in embodiment 2-7 identified with suitable functional female clone 17N16 and 17D20.The affinity maturation of these mother clones, which has obtained to survey in alternative functions, is fixed at scFv levels compared to female clone's display The kind son clone of about 2 efficiency-timed price-reforms.Son clone with optimum activity is 17N16m_d17N9 and 17D20m_d3521N11.Such as Described in embodiment 6, cloned in 17N16 mothers clone and the son of light chain rearrangement（ScFv forms, 1%NHS is determined）Functional activity In comparing, determine that 17N16m_d17N9 has than female Clone potency somewhat higher and in this measure in the son clone of all tests There is optimal function potency.

Method：

Convert and determine in the C3 carried out as described in example 5 above（1% human serum and 90% human serum）C4 conversions are neutralized to determine（90% Human serum）The comparison of the middle function potency for carrying out candidate scFv clones.

As a result it is shown in table 21 below.

Table 21：Leading son clone and their own female clone（It is all with scFv forms）With IC₅₀(nM) function effect The comparison of valency

As shown in upper table 21, when in 90% normal human serum in C3 measure（NHS）During middle measure, 17N16m_d17N9 With good activity, and it is higher than with other sub- Clone potencies of this form.

Candidate clone to IgG4, IgG4/S228P and IgG2 form conversion

All these candidate clones, which all change into IgG4, IgG4/S228P and IgG2 form, to be used to further analyze.

SEQ ID NO:62:Encoding wild type IgG4 cDNA

SEQ ID NO:63:Wild type IgG4 polypeptides

SEQ ID NO:64:Encode IgG4 mutant S228P cDNA

SEQ ID NO:65:IgG4 mutant S228P polypeptides

SEQ ID NO:69:Encoding wild type IgG2 cDNA

SEQ ID NO:70:Wild type IgG2 polypeptides.

Table 22：The summary of candidate clone：

Test monoclonal antibody #1-6 and inhuman MASP-2 albumen（Cercopithecus aethiops（AG））The cross reaction in C3 measure Ability, to determine whether that these antibody can be used in animal model（It will be predictable for people）Middle test toxicity.Also Determined in C3b depositions and C4 is surveyed to be fixed in 90% human serum and tested monoclonal antibody #1-6.As a result it is shown in table 23 below.

Table 23：The anti-MASP-2MoAb of people in 90% human serum（IC₅₀nM）

Figure 11 A illustrations MASP-2 monoclonal antibody mother clone 17N16 anti-to derived from human clone's isotype becomes Body（MoAb#1-3）The result that the C3b depositions of progress are determined.

Figure 11 B illustrations MASP-2 monoclonal antibody mother clone 17D20 anti-to derived from human clone's isotype becomes Body（MoAb#4-6）The result that the C3b depositions of progress are determined；

As shown in table 23 and Figure 11 A and 11B, the anti-MASP-2 monoclonal antibodies of people（MoAb#1-6）With high-affinity combination MASP- 2, and active suppression C3 and C4 function in 90% human serum.It is also noted that the anti-MASP-2MoAb of people and inhuman MASP-2 eggs In vain（Cercopithecus aethiops）Cross reaction, it provides the animal model for toxicity research（It will be predictable for people）.

MoAb#1-6 is further analyzed in 95% human serum and 95% cercopithecus aethiops serum.As a result it is summarised in table 24 below.

Table 24

Figure 12 A and 12B illustration deposit female clone and the MoAb#1-6 for surveying and being fixed in 95% normal human serum in C3b Test.

The suppression that C4b is deposited in 95% normal human serum of Figure 13 illustrations.

The suppression that C3b is deposited in 95% cercopithecus aethiops serum of Figure 14 illustrations.

The ability of MoAb#1-6 selective depression lectin pathways is further tested, by determining rat C3b suppression, in advance The suppression of the MBL-MASP-2 compounds of assembling, classical pathway suppresses and to C1s selectivity.As a result it is summarised in table 25.

Table 25：The summary of functional examination result

The C4 cleavage activities for the pre-assembled MBL-MASP-2 compounds that Figure 15 illustrations pass through MoAb#2,3,5 and 6 Suppress.

Figure 16 illustrations MoAb#6 compares preferential combinations of the C1s to people MASP-2.

The summary of the sequence of the son clone of table 26 in various formats：

Clone ID	Description	SEQ ID NO:
			17N16m_d17N9	Light chain gene sequence	71
17N16m_d17N9	Light chain protein sequence	72
			17N16m_d17N9	IgG2 heavy chain gene sequences	73
17N16m_d17N9	IgG2 heavy chain protein sequences	74
			17N16m_d17N9	IgG4 heavy chain gene sequences	75
17N16m_d17N9	IgG4 heavy chain protein sequences	76
			17N16m_d17N9	The heavy chain gene sequences of IgG4 mutation	77
17N17m_d17N9	The heavy chain protein sequence of IgG4 mutation	78
			17D20_3521N11	Light chain gene sequence	79
17D20_3521N11	Light chain protein sequence	80
			17D20_3521N11	IgG2 heavy chain gene sequences	81
17D20_3521N11	IgG2 heavy chain protein sequences	82
			17D20_3521N11	IgG4 heavy chain gene sequences	83
17D20_3521N11	IgG4 heavy chain protein sequences	84
			17D20_3521N11	The heavy chain gene sequences of IgG4 mutation	85
17D20_3521N11	The heavy chain protein sequence of IgG4 mutation	86
			17N16m_d17N9	The cDNA of encoding full leng scFv polypeptides	87
17D20m_d21N11	The cDNA of encoding full leng scFv polypeptides	88
			17D20m_d3521N11	The cDNA of encoding full leng scFv polypeptides	89

Embodiment 9

The epitope mapping that the description of this embodiment is carried out to the anti-MASP-2MoAb of several blocking property people.

Method：

Following recombinant protein is produced as described in example 1 above：

People MAp19

People MASP-2A

People MASP-2 SP

People MASP-2 CCP2-SP

People MASP-2 CCP1-CCP2-SP

People MASP-1/3 CUB1-EGF-CUB2

People MASP-1 CCP1-CCP2-SP.

By Dot hybridization analysis, combined on epitope, analyze anti-MASP-2 antibody OMS100 and MoAb#6（35VH- 21N11VL）, both of which be proved with high-affinity combination people MASP-2 and with block function complement activity ability （Referring to embodiment 6-8）.

Dot hybridization analysis

By on the serial dilution point sample of above-mentioned restructuring MASP-2 polypeptides to nitrocellulose filter.The amount of the albumen of point sample from 50ng to 5pg, with 10 times of steps.In later experiment, the scope of the amount of the albumen of point sample drops to 16 pg from 50ng, again with 5 times of steps Suddenly.Film is used in 5% skimmed milk powder in TBS（Block buffer）Closing, it is then slow in closing with the 1.0 anti-MASP-2Fab2 of μ g/ml Fliud flushing（Containing 5.0mMCa²⁺）It is middle to be incubated.The anti-human Fab being conjugated using HRP（AbD/Serotec;1/10,000 dilution）With ECL inspections Test agent box（Amersham）Detect the Fab2 combined.One film and the anti-human MASP-2 Ab of multi-clone rabbit（It is described in Stover Deng,J Immunol 163:6848-59 (1999)）Incubation is used as positive control.In the case, the mountain being conjugated using HRP Goat anti-rabbit igg（Dako;1/2,000 dilution）Detect the Ab combined.

As a result：

As a result it is summarised in table 27.

Table 27：Epitope mapping

As a result MoAb#6 and OMS100 antibody is shown for MASP-2 high degree of specificity and does not combine MASP-1 or MASP- 3.There is no the MASP-2 fragments of antibody binding Map19 and the CCP1 domains without MASP-2, it is binding site bag to cause conclusion Include CCP1 domains.

Embodiment 10

This embodiment proves after intravenous administration that the anti-MASP-2MoAb#6 of people suppresses the lectin pathway in cercopithecus aethiops.

Method：

MoAb#6 is to be administered to first group of three cercopithecus aethiopses and be applied with 3mg/kg dosage in 1mg/kg dose intravenous Second group to three cercopithecus aethiopses.Using 2, obtain blood sample after 4,8,10,24,48,72 and 98 hours and test whether There is lectin pathway activity.

As shown in figure 17, lectin pathway is totally constrained after the anti-human MoAb#6 of intravenous administration.

Embodiment 11

Such as this embodiment proves MASP-2 inhibitor, anti-MASP-2 antibody, treatment for radioactive exposure and/or for acute The treating, ameliorating or preventing of radiation syndrome are effective.

General principle：

Death is caused by two main mechanisms to the exposure of the ionising radiation of high dose：Bone marrow toxicity and gastrointestinal syndrome.Bone Marrow toxicity causes all blood cells to decline, and it is dead to induce body by infection and bleeding.Gastrointestinal syndrome it is more serious and By being lost and the forfeiture driving of enteroendocrine function due to the intestinal barrier function that intestinal epithelial cell layer disintegrates.This causes septicemia And related systemic inflammatorome response syndrome, it can cause death.

The lectin pathway of complement is to trigger response tissue damage and exposed to outer surface（That is bacterium）Inflammation it is congenital Immunologic mechanism.The blocking of this approach causes more preferable knot in the mouse model of ischemic intestinal tissue injury or septic shock Really.It is assumed that lectin pathway can trigger the excessive and harmful inflammation in response to radiation-induced tissue damage.Cause This, the blocking of lectin pathway can reduce secondary lesion, and increase the existence of acute radiation exposure.

The target of this research carried out described in such embodiment is by applying anti-mouse MASP-2 antibody in radiation injury Mouse model in evaluate lectin pathway and block to the effect of survival.

Study #1

Method and material：

The test article that material is used in our current research is（i）The anti-mouse MASP-2 antibody of high-affinity（mAbM11）And（ii）High parent With the anti-human MASP-2 antibody of power（mAbOMS646）, it blocks the agglutinin complement way produced in the mammalian cell of transfection The MASP-2 protein ingredients in footpath.Administration concentration is 1mg/kg anti-mouse MASP-2 antibody（mAbM11）, 5mg/kg anti-human MASP- 2 antibody（mAbOMS646）Or Sterile Saline.For each administration phase, the fresh to drug solns of enough volumes is prepared.

Animal.Young adult male Swiss- is obtained from Harlan Laboratories (Houston, TX) Webster mouse.Animal feeding in the solid bottom cage with Alpha-Dri beddings and provide certification PMI5002 rodents move Thing feed（Animal Specialties, Inc., Hubbard OR）And free water.Monitoring temperature and accommodate animal Room allows the dark light cycle in 12 hours illumination/12 hour.

Radiation.Tamed in facility after 2 weeks, 10 groups of mouse are by systemic exposure with the dose rate radiation of 0.78Gy/ minutes 6.5,7.0 and 8.0Gy, uses outfit 320-kV high-stability X-rays generator, metal-ceramic X-ray tube, variable x-ray beam Collimater and filter（Precision X-ray Incorporated, East Haven, CT）Therapax X-RAD 320 systems.

Medicine is prepared and applied.According to scheme, the Stock solutions chilled saline of the concentration of proper volume dilutes to prepare The anti-mouse MASP-2 antibody of 0.2mg/ml（mAbM11）Or the anti-human MASP-2 antibody of 0.5mg/ml（mAbOMS646）.Based on animal body Anti- MASP-2 antibody mAbM11 and mAbOMS646 are applied via IP injections using No. 25 syringe needles again, to deliver 1 mg/kg MAbM11,5mg/kg mAbOMS646 or salt solution medium.

Research and design.Mouse is assigned randomly in group as set forth in table 28.Measure daily and record body weight and body temperature.Group 7th, the mouse in 11 and 13 is put to death after irradiation and collects blood by cardiac puncture under deep anaesthesia on the 7th day.After radiation The animal of survival in 30th day is put to death and collects blood in the same way.Blood plasma is prepared simultaneously from the blood sample of collection according to scheme Being returned to sponsor is used to analyze.

Table 28：Seminar

Statistical analysis.Generation kaplan-Meier survival rate simultaneously is used to compare mean survival time between treatment group, uses Log- sorts and Wilcoxon methods.Average or average value with average standard error of the report with standard deviation.Use Double unpaired t of tail are examined carries out statistics comparison between controlled Radiata and indivedual process group.

Study #1 result

The blue Meyer survival of Kapp for 6.5,7.0 and 8.0Gy exposure groups is each provided in Figure 18 A, 18B and 18C to paint Figure, and be summarised in table 29 below.Generally, the control-animal of the radiation handled compared to medium, 6.5（20% increase）With 7.0Gy（30% increase）On exposure level, with anti-mouse MASP-2 Ab before radiation（mAbM11）Processing adds depositing for radiation murine It is living.In 6.5Gy exposure levels, radiating post processing with anti-mouse MASP-2 Ab causes the survival compared to vehicle control Radiata Rate is increased slightly（15%）.

By contrast, all processing of 7.0Gy with 8.0Gy exposure levels animal show compare medium processing spoke The survival increase for the control-animal penetrated.The maximum change of survival occurs in mAbOMS646 animal is received, sudden and violent compared to 7.0Gy The flat control-animal survival increase by 45% of dew, and in 8.0Gy exposure levels survival increase by 12%.Also, expose water in 7.0Gy Flat, the death in mAbOMS646 treatment groups is first occurred at after radiation the 15th day, and the control compared to the radiation of medium processing is moved Thing the 8th day after irradiation, is added 7 days than control-animal.In 7.0Gy exposure levels, for receiving mAbOMS646 (27.3 ± 1.3 days) dead mouse average time compare control-animal（20.7 ± 2.0 days）Dramatically increase（p = 0.0087）.

Record was compared to the radiation day before yesterday during whole research（- 1st day）The percentage change of body weight.In the dynamic of all radiation Of short duration weight loss occurs in thing, compared to control not due to the evidence of the mAbM11 or mAbOMS646 the change of divergence handled （Data are not shown）.At the end of research, the animal of all purifying is shown from starting（- 1st day）The increased weight of body weight.

Table 29：Exposed to the survival rate of the test animal of radiation

* P=0.0087, is examined by the unpaired t of double tails, the animal in control radiation and the place in identical radioactive exposure level Between reason group.

Discuss

Acute radiation syndrome is made up of the sub- symptom of three determinations：Hematopoiesis, intestines and stomach, the cerebrovascular.The symptom of observation is depended on Dose of radiation, 1Gy hematopoietic effect is exceeded with the significant locally or systemically radioactive exposure observed in people.Hematopoiesis is integrated Levy and be characterised by that the serious suppression of marrow function causes whole blood trace elements, with occurring with the infringement to immune system simultaneously In blood count, red and white haemocyte, and the change in blood platelet.When minimum point occurs, there is a small amount of neutral grain thin Born of the same parents and blood platelet are present in peripheral blood, and Neutrophilic granulocytopenia, heating, the complication of septicemia and uncontrollable bleeding cause extremely Die.

In our current research, find to apply mAbH6 increases in the Swiss-Webster male mices radiated with 7.0Gy entirely The survivability of body radiation, x-ray.It is worth noting that, in 7.0Gy exposure levels, 80% animal for receiving mAbOMS646 survival To 30 days, the control Radiata of the medium processing compared to 35%.Importantly, first day dead in this treatment group does not have There is generation, until the 15th day after radiation, compared to the increase observed in the control Radiata that medium is handled 7 days.Curiously, Exposed in lower X-ray（6.5Gy）, mAbOMS646 administration seems not compared to the animal for the control radiation that medium is handled Influence survival or delay are dead.Difference for this response between exposure level can be many-sided reason, although any The checking of hypothesis can need extra research, including mid-term sample collection and hematologic parameter for microculture.One Explain that the number of animals that can be only assigned to each group can exclude the difference for seeing that any trickle processing is related.For example, For the group size of n=20,65%（MAbOMS646, in 6.5Gy exposures）With 80%（MAbH6, in 7.0Gy exposures）Between deposit Difference living is 3 animals.On the other hand, 35%（Vehicle control, in 7.0Gy exposures）With 80%（MAbOMS646, in 7.0Gy Exposure）Between difference be 9 animals, and there is provided the admissible evidence of the related difference of processing.

These results prove, anti-MASP-2 antibody risk of the treatment with acute radiation syndrome adverse effect or by It is effective in the mammalian subject of acute radiation syndrome adverse effect.

Study #2

Swiss Webster mouse（n=50）Exposed to ionising radiation（8.0Gy）.It has rated before radioactive exposure 18 hours and afterwards The anti-MASP-2 Antybody therapies applied and applied weekly afterwards for 2 hours（OMS646 5mg/kg）To the effect of the death rate.

Study #2 result

Being defined as applying anti-MASP-2 antibody OMS646 increases the survival exposed to 8.0Gy mouse, compared to receiving vehicle control Mouse there is middle position survival rate from the adjustment of 4 to 6 days, and death subtracts when compared with the mouse for receiving vehicle control Few 12%（Log- rank tests, p=0.040）.

Study #3

Swiss Webster mouse in following group（n=50）Exposed to ionising radiation（8.0Gy）：（I：Medium）Saline control； Applied before irradiation with 2 hours after radiation within 18 hours（II：It is low）Anti- MASP-2 antibody OMS646（5mg/kg), radiate first 18 hours Applied with 2 hours after radiation（III：It is high）OMS646（10mg/kg) and only after irradiation 2 hours apply（IV:Gao Hou） OMS646（10mg/kg).

Study #3 result

The anti-MASP-2 antibody of administration is with adjusting Average Survival 4-5 before and after radiation is determined compared with the animal for receiving vehicle control My god.Death reduces 6-12% compared with vehicle control mouse in the mouse of anti-MASP-2 antibody processing.It is further noted that not having It was observed that being significantly harmful to treatment effect.

In a word, the result in the present embodiment is proved, anti-MASP-2 antibody of the invention in treatment there is acute radiation to integrate It is effective to levy in the risk of adverse effect or the mammalian subject by acute radiation syndrome adverse effect.

Embodiment 12

This embodiment further describes OMS646 antibody（17D20m_d3521N11）, i.e., have the full people of mutation anti-in hinge area The sign of people's MASP-2 IgG4 antibody.

Method：

The anti-human MASP-2 IgG4 antibody of full people that there is mutation in hinge area, OMS646 are generated as described in foregoing embodiments 2-8 Antibody（17D20m_d3521N11）.From the CHO expression cells transfected with the expression construct of coding schedule up to OMS646 weights and light chain The culture supernatants purifying OMS646 antibody of system.Cell grown cultures 16 to 20 days and works as cell in PF-CHO culture mediums Viability collects cell-free supernatants when descending below 50%.Ion exchange concentration is followed by by albumin A affinity chromatography gentle Fliud flushing exchanges to PBS purifying OMS646.

1. OMS646 is with high-affinity combination people MASP-2

The OMS646 combination recombined humans MASP-2 of immobilization surface plasma body resonant vibration（Biocore）Analysis

Method：

OMS646 with different densities by amine coupling to CM5 chip immobilizations, and with time record with 9nM, 3nM, 1nM or 0.3nM dissolving recombined human MASP-2 association and dissociation with determine combine（K_on）And dissociation（K_off）Rate constant.Based on experimental K_onAnd K_offIt is worth calculated equilibrium binding constant（K_D）.

As a result：

Figure 19 illustration surface plasma resonances（Biacore）The result of analysis, shows the OMS646 of immobilization with about 1- 3x10^-4S^-1K_offRate and about 1.6-3x10⁶M^-1S^-1K_onRate combines restructuring MASP-2, indicates affine under these experiment conditions Power（About 92pM K_D）.Depending on the concentration of MASP-2 in the OMS646 of immobilization density and solution, 50-250pM models are determined The experiment K enclosed_DValue.

The recombined human MASP-2 of OMS646 combination immobilizations ELISA is determined

Method：ELISA is carried out to determine with the dose response for the restructuring MASP-2 for evaluating OMS646 combination immobilizations.Recombined human MASP-2（50ng/ holes）It is immobilized in the maxisorpELISA plates in PBS（Nunc）On 4 DEG C overnight.Second day, plate passed through Use PBS- tweens（0.05%）Clean 3 times and closed.Then it is dilute to the coated hole addition series in Block buffer of MASP-2 The OMS646 series released（Concentration range 0.001-10nM）.It was incubated to allow the antigen of OMS646 combination immobilizations by 1 hour Afterwards, clean-out opening is to remove uncombined OMS646.The goat anti-human IgG antibodies marked using HRP（Qualex；1:5000 dilutions In Block buffer）Then use TMB peroxidase substrates（Kirkegaard & Perry Laboratories）Colour developing inspection Survey the OMS646 combined.By adding the M H of 100 μ l/ holes 1.0₃PO₄Terminate peroxidase reaction and substrate conversion is used 96 plate readers（Spectramax）It is quantitative in 450nM light measurements.Use the curve fitting algorithm of single binding site （Graphpad）To calculate K_DValue.

As a result：

The result that Figure 20 illustrations ELISA is determined is to determine binding affinities of the OMS646 to the people MASP-2 of immobilization.Such as Shown in Figure 20, it is determined that OMS646 is with 85 ± 5 pM K_DWith reference to the recombined human MASP-2 of immobilization, its with it is as shown in figure 19 The result obtained in Biocore analyses is consistent.These results prove that OMS646 has high-affinity to people MASP-2, with about 100pM K_D。

2. OMS646 suppresses not press down on C4 activation but the coated surface of immune complex on the coated surface of mannosan System

Method：

Respectively on the coated surface of mannosan or the coated surface of immune complex in the situation presence or absence of OMS646 Under shown in Figure 21 A and 21B concentration range measurement C4 activation.

In the method for following measurement MASP-2 C4 cleavage activities formation, the plastic eyelet of mannosan is coated with 37 DEG C 60 minutes are incubated together with 1% human serum to activate lectin pathway.Then clean-out opening and determined using standard ELISA assay It is immobilized in the people C4b on hole.This C4b generated in determining amount is measuring for the C4 cleavage activities that MASP-2 is relied on.This is surveyed The ability in their the suppression C4 cuttings of the anti-MASP-2 antibody of selected concentration is tested in fixed.

Method：

C4 activation on the coated surface of mannosan：

In order to determine that adhesion plate is coated with mannosan in effects of the OMS646 to lectin pathway, 96 hole Costar, is passed through At 5 DEG C 50mM carbonate buffer solutions are diluted in the μ g/mL of 50 μ L 40（pH9.5）In mannosan be incubated overnight.Used per hole 200 μ L PBSs 3 times.Then closed and gently mixed in room temperature with 1% bovine serum albumin(BSA) in the PBS in 100 μ L/ holes in hole It is incubated 1 hour.Per hole with 200 μ L PBSs 3 times.In single 96 orifice plate, MASP-2 antibody（OMS646）It is serial dilute Release with 1% human serum containing Ca⁺⁺And Mg⁺⁺GVB buffer solutions（4.0 mM barbitals, 141 mM NaCl, 1.0 mM MgCl₂、 2.0 mM CaCl₂, 0.1% gelatin, pH 7.4）In in 5 DEG C of preincubates 1 hour.These antibody-serum preincubated mixture is subsequent In the respective aperture for being transferred to the coated assay plate of mannosan.Trigger complement activation by the way that assay plate is transferred into 37 DEG C of water-baths. After 60 minutes are incubated, by adding EDTA terminating reactions to reactant mixture.Per hole PBS polysorbas20s（In PBS 0.05% polysorbas20）The μ L of 5 x 200 are cleaned, are then cleaned 2 times with 200 μ LPBS per hole.The chicken of 100 μ L/ holes biotin-conjugateds resists The 1 of people C4c (Immunsystem AB, Uppsala, Sweden)：100 dilutions are added to pure comprising 2.0mg/ml ox bloods Albumen（BSA）PBS in and at room temperature gentle mixing be incubated 1 hour.Per the hole μ L of 5 x of PBS 200.100 μ L/ holes The Streptavidin (Pierce Chemical #21126) of 0.1 μ g/ml peroxidase conjugated, which is added to, to be included Gentle mixing is incubated 1 hour in 2.0mg/mlBSA PBS and at room temperature on shaking table.Per the hole μ of 5 x of PBS 200 L.Add the peroxidase substrate TMB in 100 μ L/ holes（Kirkegaard ＆ Perry Laboratories) and incubated in room temperature Educate 10 minutes.The 1.0 M H that peroxidase reaction passes through 100 μ L/ holes of addition₃PO₄Terminate and measure OD₄₅₀。

C4 activation on the coated surface of immune complex：

In order to measure effects of the OMS646 to classical pathway, said determination is revised as using the coated plate of immune complex.It is such as right It is described in detail and is measured in above-mentioned lectin pathway, difference is that hole is coated with the sheep IgG of purifying for via classical way Footpath stimulates C4 to activate.

As a result：

Figure 21 A illustrations are on the coated surface of mannosan presence or absence of C4 activation levels in the case of OMS646. Figure 21 B illustrations are on the coated surfaces of IgG presence or absence of C4 activation levels in the case of OMS646.As in Figure 21 A Shown, OMS646 is on the coated surface of mannosan with about 0.5nM IC in 1% human serum₅₀Suppress C4 activation.At 10 The IC obtained in independent experiment₅₀Value is 0.52 ± 0.28 nM (average value ± SD).On the contrary, as illustrated in fig. 21b, OMS646 exists C4 activation is not suppressed on the coated surfaces of IgG.These results prove that OMS646 blocks the Lectin-dependent of complement component C4, but The activation that non-classical approach is relied on.

3. The activation of the Lectin-dependent of OMS646 specific inhibition terminal complement compositions

Method：

Film is attacked using the approach specific condition analysis OMS646 for lectin pathway, classical pathway and alternative route Compound（MAC）The effect of deposition.For this purpose, screened according to the explanation of manufacturer using Wieslab Comp300 complements Kit (Wieslab, Lund, Sweden).

As a result：

Figure 22 A illustrations are under the conditions of lectin pathway specific assay presence or absence of anti-MASP-2 antibody（OMS646） In the case of MAC deposit level.Figure 22 B illustrations are under the conditions of classical pathway specific assay presence or absence of anti- MASP-2 antibody（OMS646）In the case of MAC deposit level.Figure 22 C illustrations are in alternative route specific assay condition It is lower presence or absence of anti-MASP-2 antibody（OMS646）In the case of MAC deposit level.

As shown in fig. 22, OMS646 blocks the activation of the lectin pathway mediation of MAC depositions, the IC with about 1nM₅₀Value. But activation of the OMS646 to being mediated from classical pathway（Figure 22 B）Or the activation mediated from alternative route（Figure 22 C）The MAC of generation It is deposited without effect.

4. OMS646 effectively suppresses lectin pathway activation in physiological conditions

Method：

In 90% human serum presence or absence of the OMS646 of following various concentrations in the case of evaluate Lectin-dependent C3 and C4 is activated：

C4 activation determinations

It is poly- with 2 μ g sweet dews in order to evaluate adhesion plate in effects of the OMS646 to the C4 activation of Lectin-dependent, 96 hole Costar Sugar（The μ g/mL of 50 μ L 40 are in 50mM carbonate buffer solutions（pH9.5）In solution）Coating.Then plate cleans 3 with 200 μ LPBS It is secondary and with 1% bovine serum albumin(BSA) in the PBS in 100 μ L/ holes room temperature gently mix closing 1 hour.In single preincubate plate In, the OMS646 of selection concentration is mixed with 90% human serum and is incubated 1 hour on ice.These antibody-serum preincubated mixture In the hole for being subsequently transferred to the coated assay plate of mannosan on ice.Then assay plate is incubated 90 minutes to permit in ice-water bath Perhaps complement activation.By adding EDTA terminating reactions to reactant mixture.Per hole with 200 μ LPBS polysorbas20s（In PBS 0.05% polysorbas20）Cleaning 5 times, is then cleaned 2 times per hole with 200 μ LPBS.The anti-human C4c of chicken of 100 μ L/ holes biotin-conjugateds The 1 of (Immunsystem AB, Uppsala, Sweden)：1000 dilutions are added to comprising 2.0 mg/ml bovine serum albumin(BSA)s （BSA）PBS in and at room temperature gentle mixing be incubated 1 hour.Per hole with 200 μ L PBSs 5 times.The μ of 100 μ L/ holes 0.1 The Streptavidin (Pierce Chemical #21126) of g/mL peroxidase conjugated is added to comprising 2.0mg/ Gentle mixing is incubated 1 hour in mlBSA PBS and at room temperature on shaking table.Per hole with 200 μ L PBSs 5 times.Addition The peroxidase substrate TMB in 100 μ L/ holes（Kirkegaard ＆ Perry Laboratories) and in 16 points of incubation at room temperature Clock.The 1.0 M H that peroxidase reaction passes through 100 μ L/ holes of addition₃PO₄Terminate and measure OD₄₅₀。

C4 activation determinations

In order to evaluate effects of the OMS646 to the C3 activation of Lectin-dependent, to be carried out with above-mentioned C4 activation determinations identical mode Determine, except C3 is deposited as end points evaluation.C3 depositions are following quantitative.

The end reacted in complement deposit, plate is cleaned and then with the cow's serum containing 2.0mg/ml in 100 μ L/ holes as described above Albumin（BSA）PBS in rabbit-anti people's C3c antibody（Dako）1:5000 dilutions are incubated 1 hour.It is clear with 200 μ L PBS per plate Wash 5 times, and the goat anti-rabbit igg that then HRP is marked in the BSA containing 2.0mg/ml in room temperature Yu 100 μ L/ holes PBS （American Qualex Antibodies）It is incubated 1 hour.Plate is cleaned 5 times and and then 100 μ L/ holes of addition with 200 μ LPBS Peroxidase substrate TMB（Kirkegaard ＆ Perry Laboratories) and in incubation at room temperature 10 minutes.Peroxidating The 1.0 M H that thing enzyme reaction passes through 100 μ L/ holes of addition₃PO₄Terminate and measure OD₄₅₀.By to experimental data set application S-shaped agent Amount-response curve fitting algorithm（GraphPad）Derivative IC₅₀Value.

As a result：

Figure 23 A illustrations are under lectin pathway particular conditions, anti-presence or absence of a series of anti-MASP-2 of concentration Body（OMS646）In the case of in 90% human serum C3 deposit level.Figure 23 B illustrations are in the specific bar of lectin pathway Under part, presence or absence of a series of anti-MASP-2 antibody of concentration（OMS646）In the case of in 90% human serum C4 deposit Level.As shown in fig. 23 a, OMS646 blocks C3 depositions in 90% human serum, with IC₅₀= 3±1.5 nM (n=6).Such as Shown in Figure 23 B, OMS646 blocks C4 depositions, with IC₅₀ = 2.8±1.3 nM (n=6)。

These results prove that OMS646 provides the strength activated in physiological conditions to lectin pathway, is effectively blocked, Thus the use to OMS646 low therapeutic dose provides support.Based on these data it was expected that with 20 nM (3 μ g/mL) or more The OMS646 of low plasma concentration will be blocked in patient's circulation>90% lectin pathway.Typical human blood slurry based on about 3L Product, and the lot of antibodies material applied are retained in the knowledge in blood plasma（Lin Y.S. etc., JPET 288:371 (1999)）, it is contemplated that the intravenous dosage for applying low such as 10mg OMS646 will be during acute period（I.e. of short duration period, example Such as 1-3 days）It is effective during blocked lectin element approach.In the case of chronic disease, the blocked lectin in the time cycle of extension Plain approach can be favourable for realizing maximum treatment interests.Therefore, can preferably 100mg for these chronic conditions OMS646 dosage, it is contemplated that it during blocked lectin element approach at least one week or longer is effective in adult subjects.Mirror In the people of slow removing and long half-lift be generally observed in to(for) antibody, the OMS646 of possible 100mg dosage is for being longer than 1 Week, such as 2 weeks, or can be effective in even 4 weeks.It is expected that the higher doses of antibody（That is, higher than 100mg, such as 200mg, 500mg or higher, such as 700mg or 1000mg）By with longer acting duration（For example, more than 2 weeks）.

5. OMS646 blocks the lectin pathway activation in monkey

As described in Example 10 with shown in Figure 17, it is determined that to the intravenous administration OMS646 of cercopithecus aethiops（3mg/kg) Afterwards, OMS646 eliminates the whole body lectin pathway activity period of about 72 hours, subsequent lectin pathway activation recovering.

This embodiment describes follow-up investigation, wherein in 90% cercopithecus aethiops serum or in 90%Cynomoglus monkey serums The C4 activity of Lectin-dependent is evaluated in the case of to a series of OMS646 and in the absence of OMS646, it is as follows：

In order to evaluate the effect that OMS646 is activated in different non-human primate species to the C4 of Lectin-dependent, 96 holes Adhesion plate is with 2 μ g mannosans in Costar（The μ g/mL of 50 μ L 40 are in 50mM carbonate buffer solutions（pH9.5）In it is molten Liquid）Coating.Then plate is cleaned 3 times and gentle in room temperature with 1% bovine serum albumin(BSA) in the PBS in 100 μ L/ holes with 200 μ LPBS Mixing closing 1 hour.In single preincubate plate, the OMS646 of concentration is selected with being received from cercopithecus aethiops or Cynomoglus monkeys The 90% serum mixing of collection is simultaneously incubated 1 hour on ice.These antibody-serum preincubated mixture is subsequently transferred on ice sweet In the hole for revealing the coated assay plate of glycan.Then assay plate is incubated 90 minutes to allow complement activation in ice-water bath.By to Reactant mixture adds EDTA terminating reactions.Per hole with 200 μ LPBS- polysorbas20s（0.05% polysorbas20 in PBS）Cleaning 5 times, Then cleaned 2 times with 200 μ LPBS per hole.100 μ L/ holes biotin-conjugateds the anti-human C4c of chicken (Immunsystem AB, Uppsala, Sweden) 1：1000 dilutions are added in the PBS comprising 2.0mg/ml BSA and gentle mixing at room temperature It is incubated 1 hour.Per hole with 200 μ L PBSs 5 times.The strepto- of the μ g/mL of 100 μ L/ holes 0.1 peroxidase conjugated is affine Plain (Pierce Chemical #21126) is added in the PBS comprising 2.0mg/mlBSA and gentle on shaking table at room temperature Mixing is incubated 1 hour.Per hole with 200 μ L PBSs 5 times.Add the peroxidase substrate TMB in 100 μ L/ holes （Kirkegaard ＆ Perry Laboratories) and in incubation at room temperature 10 minutes.Peroxidase reaction passes through addition 100 The 1.0 M H in μ L/ holes₃PO₄Terminate and measure OD₄₅₀.By to experimental data set application S-shaped dosage-response curve fitting algorithm （GraphPad）Derivative IC₅₀Value.

As a result：

In 90%Cynomoglus monkey serums（Figure 24 A）With 90% cercopithecus aethiops serum（Figure 24 B）In observe lectin pathway suppression The dose response of system, the respectively IC with 30nM-50nM and 15 nM-30 nM scopes₅₀Value.

In a word, it was observed that OMS646, the complete anti-human MASP-2 IGG4 antibody of people（There is mutation in hinge area）With following Favorable property：With reference to people MASP-2 high-affinity（The K of 50-250 pM scopes_D, 1-3X10^-4 S^-1The K of scope_offRate and 1.6- 3X10⁶M^-1S^-1The K of scope_onRate；The suppression that function potency in human serum is deposited for C4, has 0.52 in 1% human serum ± 0.28 nM (N=10) IC₅₀；And there is 3 ± 1.5nM IC in 90% serum₅₀）；With the cross reaction in monkey Property, the suppression of display C4 depositions, the IC with 15-50nM scopes₅₀（90% monkey serum）..

As described above, it is contemplated that low such as 10mg OMS646 dosage（For average people correspondence 0.15mg/kg）Followed in acute blocking people During lectin pathway in ring（For example at least period of 1-3 days）It is effective, and is expected 100mg OMS646 dosage （For average people correspondence 1.5mg/kg）Block the lectin pathway at least 1 week or longer in patient's circulation.It can use bigger The OMS646 of dosage（For example, the dosage higher than 100mg, for example, at least 200mg, at least 300mg, at least 400mg, at least 500mg Or it is higher）And it is preferably subcutaneous（sc）Or it is intramuscular（im）Route of administration is further to extend the time that effective lectin pathway is eliminated Window is to 2 weeks and preferably 4 weeks.

For example, as shown in this paper experimental datas, in primate, OMS646 1mg/kg dosage causes agglutinin way Footpath suppresses 1 day, and OMS646 3mg/kg dosage causes the suppression about 3 days of lectin pathway（72 hours）.Therefore, 7- is estimated 10mg/kg higher doses suppresses the lectin pathway period of about 7 days by effective.As shown here, OMS646 compares monkey MASP- 2 have potency 5-10 times higher for people MASP-2.It is assumed that suitable pharmacokinetics, realizes effective agglutinin in people The projected dose scope that approach is eliminated is shown in table 30 below.

Table 30：Suppress the OMS646 dosage of internal lectin pathway

	1 day	3 days	7 days
				Monkey	1 mg/kg	3 mg/kg	10 mg/kg
People（Estimation）	0.1-0.2 mg/kg	0.3-0.6 mg/kg	1-2 mg/kg

Although the preferred embodiments of the invention have been illustrated and described, it will recognize wherein carry out various changes Without departing from the spirit and scope of the present invention.

Sequence table

<110> Omeros Corporation

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Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr Pro Met

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Ala Pro Pro Asn Gly His Val Ser Pro Val Gln Ala Lys Tyr Ile Leu

305 310 315 320

Lys Asp Ser Phe Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu Gln

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Gly His Leu Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly

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Ser Trp Asp Arg Pro Met Pro Ala Cys Ser Ile Val Asp Cys Gly Pro

355 360 365

Pro Asp Asp Leu Pro Ser Gly Arg Val Glu Tyr Ile Thr Gly Pro Gly

370 375 380

Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe

385 390 395 400

Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr Val Cys Glu Ala Asp Gly

405 410 415

Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Glu Pro

420 425 430

Val Cys Gly Leu Ser Ala Arg Thr Thr Gly Gly Arg Ile Tyr Gly Gly

435 440 445

Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Ile Leu Gly

450 455 460

Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr Asp Asn Trp Val Leu Thr

465 470 475 480

Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala Ser Ala Leu Asp

485 490 495

Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His Tyr Thr Gln Ala

500 505 510

Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Asp Ala Gly

515 520 525

Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn Lys Val Val Ile

530 535 540

Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys Glu Ala Glu Ser

545 550 555 560

Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly Trp Gly Leu Thr

565 570 575

Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val Asp Ile Pro Ile

580 585 590

Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys Pro Pro Tyr Pro

595 600 605

Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly Leu Glu Ser Gly

610 615 620

Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu

625 630 635 640

Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly

645 650 655

Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val

660 665 670

Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser Asp Phe

675 680 685

<210> 3

<211> 671

<212> PRT

<213>Homo sapiens

<400> 3

Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala

1 5 10 15

Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp

20 25 30

Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His

35 40 45

Phe Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu

50 55 60

Ser Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr

65 70 75 80

Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser

85 90 95

Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe

100 105 110

Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln

115 120 125

Val Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His

130 135 140

Leu Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg

145 150 155 160

Asn Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln

165 170 175

Arg Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg Pro Tyr Pro Lys

180 185 190

Leu Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Glu Gly Phe Ser Val

195 200 205

Ile Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr

210 215 220

Leu Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His

225 230 235 240

Gly Pro Phe Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser

245 250 255

Asn Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr

260 265 270

Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr Pro

275 280 285

Met Ala Pro Pro Asn Gly His Val Ser Pro Val Gln Ala Lys Tyr Ile

290 295 300

Leu Lys Asp Ser Phe Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu

305 310 315 320

Gln Gly His Leu Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp

325 330 335

Gly Ser Trp Asp Arg Pro Met Pro Ala Cys Ser Ile Val Asp Cys Gly

340 345 350

Pro Pro Asp Asp Leu Pro Ser Gly Arg Val Glu Tyr Ile Thr Gly Pro

355 360 365

Gly Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr

370 375 380

Phe Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr Val Cys Glu Ala Asp

385 390 395 400

Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Glu

405 410 415

Pro Val Cys Gly Leu Ser Ala Arg Thr Thr Gly Gly Arg Ile Tyr Gly

420 425 430

Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Ile Leu

435 440 445

Gly Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr Asp Asn Trp Val Leu

450 455 460

Thr Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala Ser Ala Leu

465 470 475 480

Asp Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His Tyr Thr Gln

485 490 495

Ala Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Asp Ala

500 505 510

Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn Lys Val Val

515 520 525

Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys Glu Ala Glu

530 535 540

Ser Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly Trp Gly Leu

545 550 555 560

Thr Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val Asp Ile Pro

565 570 575

Ile Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys Pro Pro Tyr

580 585 590

Pro Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly Leu Glu Ser

595 600 605

Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe

610 615 620

Leu Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile Val Ser Trp

625 630 635 640

Gly Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val Tyr Thr Lys

645 650 655

Val Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser Asp Phe

660 665 670

<210> 4

<211> 2091

<212> DNA

<213>Rat

<220>

<221> CDS

<222> (10)..(2067)

<400> 4

tggcacaca atg agg cta ctg atc gtc ctg ggt ctg ctt tgg agt ttg gtg 51

Met Arg Leu Leu Ile Val Leu Gly Leu Leu Trp Ser Leu Val

1 5 10

gcc aca ctt ttg ggc tcc aag tgg cct gag cct gta ttc ggg cgc ctg 99

Ala Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu

15 20 25 30

gtg tcc ctg gcc ttc cca gag aag tat ggc aac cat cag gat cga tcc 147

Val Ser Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser

35 40 45

tgg acg ctg act gca ccc cct ggc ttc cgc ctg cgc ctc tac ttc acc 195

Trp Thr Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr

50 55 60

cac ttc aac ctg gaa ctc tct tac cgc tgc gag tat gac ttt gtc aag 243

His Phe Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys

65 70 75

ttg acc tca ggg acc aag gtg cta gcc acg ctg tgt ggg cag gag agt 291

Leu Thr Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser

80 85 90

aca gat act gag cgg gca cct ggc aat gac acc ttc tac tca ctg ggt 339

Thr Asp Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly

95 100 105 110

ccc agc cta aag gtc acc ttc cac tcc gac tac tcc aat gag aag cca 387

Pro Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro

115 120 125

ttc aca gga ttt gag gcc ttc tat gca gcg gag gat gtg gat gaa tgc 435

Phe Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys

130 135 140

aga aca tcc ctg gga gac tca gtc cct tgt gac cat tat tgc cac aac 483

Arg Thr Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn

145 150 155

tac ctg ggc ggc tac tac tgc tcc tgc cga gtg ggc tac att ctg cac 531

Tyr Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His

160 165 170

cag aac aag cat acc tgc tca gcc ctt tgt tca ggc cag gtg ttc act 579

Gln Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr

175 180 185 190

ggg agg tct ggc ttt ctc agt agc cct gag tac cca cag cca tac ccc 627

Gly Arg Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro

195 200 205

aaa ctc tcc agc tgc gcc tac aac atc cgc ctg gag gaa ggc ttc agt 675

Lys Leu Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser

210 215 220

atc acc ctg gac ttc gtg gag tcc ttt gat gtg gag atg cac cct gaa 723

Ile Thr Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu

225 230 235

gcc cag tgc ccc tac gac tcc ctc aag att caa aca gac aag agg gaa 771

Ala Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu

240 245 250

tac ggc ccg ttt tgt ggg aag acg ctg ccc ccc agg att gaa act gac 819

Tyr Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp

255 260 265 270

agc aac aag gtg acc att acc ttt acc acc gac gag tca ggg aac cac 867

Ser Asn Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His

275 280 285

aca ggc tgg aag ata cac tac aca agc aca gca cag ccc tgc cct gat 915

Thr Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp

290 295 300

cca acg gcg cca cct aat ggt cac att tca cct gtg caa gcc acg tat 963

Pro Thr Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr

305 310 315

gtc ctg aag gac agc ttt tct gtc ttc tgc aag act ggc ttc gag ctt 1011

Val Leu Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu

320 325 330

ctg caa ggt tct gtc ccc ctg aag tca ttc act gct gtc tgt cag aaa 1059

Leu Gln Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys

335 340 345 350

gat gga tct tgg gac cgg ccg ata cca gag tgc agc att att gac tgt 1107

Asp Gly Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Ile Asp Cys

355 360 365

ggc cct ccc gat gac cta ccc aat ggc cac gtg gac tat atc aca ggc 1155

Gly Pro Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly

370 375 380

cct gaa gtg acc acc tac aaa gct gtg att cag tac agc tgt gaa gag 1203

Pro Glu Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu

385 390 395

act ttc tac aca atg agc agc aat ggt aaa tat gtg tgt gag gct gat 1251

Thr Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp

400 405 410

gga ttc tgg acg agc tcc aaa gga gaa aaa tcc ctc ccg gtt tgc aag 1299

Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys

415 420 425 430

cct gtc tgt gga ctg tcc aca cac act tca gga ggc cgt ata att gga 1347

Pro Val Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly

435 440 445

gga cag cct gca aag cct ggt gac ttt cct tgg caa gtc ttg tta ctg 1395

Gly Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu

450 455 460

ggt gaa act aca gca gca ggt gct ctt ata cat gac gac tgg gtc cta 1443

Gly Glu Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asp Trp Val Leu

465 470 475

aca gcg gct cat gct gta tat ggg aaa aca gag gcg atg tcc tcc ctg 1491

Thr Ala Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu

480 485 490

gac atc cgc atg ggc atc ctc aaa agg ctc tcc ctc att tac act caa 1539

Asp Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln

495 500 505 510

gcc tgg cca gag gct gtc ttt atc cat gaa ggc tac act cac gga gct 1587

Ala Trp Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala

515 520 525

ggt ttt gac aat gat ata gca ctg att aaa ctc aag aac aaa gtc aca 1635

Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr

530 535 540

atc aac aga aac atc atg ccg att tgt cta cca aga aaa gaa gct gca 1683

Ile Asn Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala

545 550 555

tcc tta atg aaa aca gac ttc gtt gga act gtg gct ggc tgg ggg tta 1731

Ser Leu Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu

560 565 570

acc cag aag ggg ttt ctt gct aga aac cta atg ttt gtg gac ata cca 1779

Thr Gln Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro

575 580 585 590

att gtt gac cac caa aaa tgt gct act gcg tat aca aag cag ccc tac 1827

Ile Val Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr

595 600 605

cca gga gca aaa gtg act gtt aac atg ctc tgt gct ggc cta gac cgc 1875

Pro Gly Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg

610 615 620

ggt ggc aag gac agc tgc aga ggt gac agc gga ggg gca tta gtg ttt 1923

Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe

625 630 635

cta gac aat gaa aca cag aga tgg ttt gtg gga gga ata gtt tcc tgg 1971

Leu Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp

640 645 650

ggt tct att aac tgt ggg ggg tca gaa cag tat ggg gtc tac acg aaa 2019

Gly Ser Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys

655 660 665 670

gtc acg aac tat att ccc tgg att gag aac ata ata aat aat ttc taa 2067

Val Thr Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe

675 680 685

tttgcaaaaa aaaaaaaaaa aaaa 2091

<210> 5

<211> 685

<212> PRT

<213>Rat

<400> 5

Met Arg Leu Leu Ile Val Leu Gly Leu Leu Trp Ser Leu Val Ala Thr

1 5 10 15

Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val Ser

20 25 30

Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser Trp Thr

35 40 45

Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr His Phe

50 55 60

Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu Thr

65 70 75 80

Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp

85 90 95

Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro Ser

100 105 110

Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr

115 120 125

Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg Thr

130 135 140

Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr Leu

145 150 155 160

Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His Gln Asn

165 170 175

Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly Arg

180 185 190

Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys Leu

195 200 205

Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser Ile Thr

210 215 220

Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu Ala Gln

225 230 235 240

Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu Tyr Gly

245 250 255

Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser Asn

260 265 270

Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His Thr Gly

275 280 285

Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp Pro Thr

290 295 300

Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr Val Leu

305 310 315 320

Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu Gln

325 330 335

Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly

340 345 350

Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Ile Asp Cys Gly Pro

355 360 365

Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro Glu

370 375 380

Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe

385 390 395 400

Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly Phe

405 410 415

Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys Pro Val

420 425 430

Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly Gly Gln

435 440 445

Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly Glu

450 455 460

Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asp Trp Val Leu Thr Ala

465 470 475 480

Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu Asp Ile

485 490 495

Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln Ala Trp

500 505 510

Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala Gly Phe

515 520 525

Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr Ile Asn

530 535 540

Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala Ser Leu

545 550 555 560

Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu Thr Gln

565 570 575

Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro Ile Val

580 585 590

Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr Pro Gly

595 600 605

Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg Gly Gly

610 615 620

Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu Asp

625 630 635 640

Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly Ser

645 650 655

Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys Val Thr

660 665 670

Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe

675 680 685

<210> 6

<211> 670

<212> PRT

<213>Rat

<400> 6

Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val

1 5 10 15

Ser Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser Trp

20 25 30

Thr Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr His

35 40 45

Phe Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu

50 55 60

Thr Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr

65 70 75 80

Asp Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro

85 90 95

Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe

100 105 110

Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg

115 120 125

Thr Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr

130 135 140

Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His Gln

145 150 155 160

Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly

165 170 175

Arg Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys

180 185 190

Leu Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser Ile

195 200 205

Thr Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu Ala

210 215 220

Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu Tyr

225 230 235 240

Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser

245 250 255

Asn Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His Thr

260 265 270

Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp Pro

275 280 285

Thr Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr Val

290 295 300

Leu Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu

305 310 315 320

Gln Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp

325 330 335

Gly Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Ile Asp Cys Gly

340 345 350

Pro Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro

355 360 365

Glu Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr

370 375 380

Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly

385 390 395 400

Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys Pro

405 410 415

Val Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly Gly

420 425 430

Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly

435 440 445

Glu Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asp Trp Val Leu Thr

450 455 460

Ala Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu Asp

465 470 475 480

Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln Ala

485 490 495

Trp Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala Gly

500 505 510

Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr Ile

515 520 525

Asn Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala Ser

530 535 540

Leu Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu Thr

545 550 555 560

Gln Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro Ile

565 570 575

Val Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr Pro

580 585 590

Gly Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg Gly

595 600 605

Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu

610 615 620

Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly

625 630 635 640

Ser Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys Val

645 650 655

Thr Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe

660 665 670

<210> 7

<211> 136

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 7

Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr

1 5 10 15

Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser

20 25 30

Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr

35 40 45

Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe

50 55 60

Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser

65 70 75 80

Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp

85 90 95

Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser

100 105 110

Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr

115 120 125

Gly Phe Glu Ala Phe Tyr Ala Ala

130 135

<210> 8

<211> 181

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 8

Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr

1 5 10 15

Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser

20 25 30

Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr

35 40 45

Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe

50 55 60

Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser

65 70 75 80

Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp

85 90 95

Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser

100 105 110

Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr

115 120 125

Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val

130 135 140

Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu

145 150 155 160

Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn

165 170 175

Lys Arg Thr Cys Ser

180

<210> 9

<211> 277

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 9

Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala

1 5 10 15

Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp

20 25 30

Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His

35 40 45

Phe Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu

50 55 60

Ser Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr

65 70 75 80

Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser

85 90 95

Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe

100 105 110

Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln

115 120 125

Val Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His

130 135 140

Leu Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg

145 150 155 160

Asn Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln

165 170 175

Arg Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg Pro Tyr Pro Lys

180 185 190

Leu Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Glu Gly Phe Ser Val

195 200 205

Ile Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr

210 215 220

Leu Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His

225 230 235 240

Gly Pro Phe Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser

245 250 255

Asn Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr

260 265 270

Gly Trp Lys Ile His

275

<210> 10

<211> 41

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 10

Glu Asp Ile Asp Glu Cys Gln Val Ala Pro Gly Glu Ala Pro Thr Cys

1 5 10 15

Asp His His Cys His Asn His Leu Gly Gly Phe Tyr Cys Ser Cys Arg

20 25 30

Ala Gly Tyr Val Leu His Arg Asn Lys

35 40

<210> 11

<211> 394

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 11

Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr Pro Met Ala Pro Pro Asn

1 5 10 15

Gly His Val Ser Pro Val Gln Ala Lys Tyr Ile Leu Lys Asp Ser Phe

20 25 30

Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu Gln Gly His Leu Pro

35 40 45

Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly Ser Trp Asp Arg

50 55 60

Pro Met Pro Ala Cys Ser Ile Val Asp Cys Gly Pro Pro Asp Asp Leu

65 70 75 80

Pro Ser Gly Arg Val Glu Tyr Ile Thr Gly Pro Gly Val Thr Thr Tyr

85 90 95

Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe Tyr Thr Met Lys

100 105 110

Val Asn Asp Gly Lys Tyr Val Cys Glu Ala Asp Gly Phe Trp Thr Ser

115 120 125

Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Glu Pro Val Cys Gly Leu

130 135 140

Ser Ala Arg Thr Thr Gly Gly Arg Ile Tyr Gly Gly Gln Lys Ala Lys

145 150 155 160

Pro Gly Asp Phe Pro Trp Gln Val Leu Ile Leu Gly Gly Thr Thr Ala

165 170 175

Ala Gly Ala Leu Leu Tyr Asp Asn Trp Val Leu Thr Ala Ala His Ala

180 185 190

Val Tyr Glu Gln Lys His Asp Ala Ser Ala Leu Asp Ile Arg Met Gly

195 200 205

Thr Leu Lys Arg Leu Ser Pro His Tyr Thr Gln Ala Trp Ser Glu Ala

210 215 220

Val Phe Ile His Glu Gly Tyr Thr His Asp Ala Gly Phe Asp Asn Asp

225 230 235 240

Ile Ala Leu Ile Lys Leu Asn Asn Lys Val Val Ile Asn Ser Asn Ile

245 250 255

Thr Pro Ile Cys Leu Pro Arg Lys Glu Ala Glu Ser Phe Met Arg Thr

260 265 270

Asp Asp Ile Gly Thr Ala Ser Gly Trp Gly Leu Thr Gln Arg Gly Phe

275 280 285

Leu Ala Arg Asn Leu Met Tyr Val Asp Ile Pro Ile Val Asp His Gln

290 295 300

Lys Cys Thr Ala Ala Tyr Glu Lys Pro Pro Tyr Pro Arg Gly Ser Val

305 310 315 320

Thr Ala Asn Met Leu Cys Ala Gly Leu Glu Ser Gly Gly Lys Asp Ser

325 330 335

Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu Asp Ser Glu Thr

340 345 350

Glu Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly Ser Met Asn Cys

355 360 365

Gly Glu Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val Ile Asn Tyr Ile

370 375 380

Pro Trp Ile Glu Asn Ile Ile Ser Asp Phe

385 390

<210> 12

<211> 152

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 12

Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr Pro Met Ala Pro Pro Asn

1 5 10 15

Gly His Val Ser Pro Val Gln Ala Lys Tyr Ile Leu Lys Asp Ser Phe

20 25 30

Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu Gln Gly His Leu Pro

35 40 45

Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly Ser Trp Asp Arg

50 55 60

Pro Met Pro Ala Cys Ser Ile Val Asp Cys Gly Pro Pro Asp Asp Leu

65 70 75 80

Pro Ser Gly Arg Val Glu Tyr Ile Thr Gly Pro Gly Val Thr Thr Tyr

85 90 95

Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe Tyr Thr Met Lys

100 105 110

Val Asn Asp Gly Lys Tyr Val Cys Glu Ala Asp Gly Phe Trp Thr Ser

115 120 125

Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Glu Pro Val Cys Gly Leu

130 135 140

Ser Ala Arg Thr Thr Gly Gly Arg

145 150

<210> 13

<211> 70

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 13

Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr Pro Met Ala Pro Pro Asn

1 5 10 15

Gly His Val Ser Pro Val Gln Ala Lys Tyr Ile Leu Lys Asp Ser Phe

20 25 30

Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu Gln Gly His Leu Pro

35 40 45

Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly Ser Trp Asp Arg

50 55 60

Pro Met Pro Ala Cys Ser

65 70

<210> 14

<211> 324

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 14

Ile Val Asp Cys Gly Pro Pro Asp Asp Leu Pro Ser Gly Arg Val Glu

1 5 10 15

Tyr Ile Thr Gly Pro Gly Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr

20 25 30

Ser Cys Glu Glu Thr Phe Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr

35 40 45

Val Cys Glu Ala Asp Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser

50 55 60

Leu Pro Val Cys Glu Pro Val Cys Gly Leu Ser Ala Arg Thr Thr Gly

65 70 75 80

Gly Arg Ile Tyr Gly Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp

85 90 95

Gln Val Leu Ile Leu Gly Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr

100 105 110

Asp Asn Trp Val Leu Thr Ala Ala His Ala Val Tyr Glu Gln Lys His

115 120 125

Asp Ala Ser Ala Leu Asp Ile Arg Met Gly Thr Leu Lys Arg Leu Ser

130 135 140

Pro His Tyr Thr Gln Ala Trp Ser Glu Ala Val Phe Ile His Glu Gly

145 150 155 160

Tyr Thr His Asp Ala Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu

165 170 175

Asn Asn Lys Val Val Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro

180 185 190

Arg Lys Glu Ala Glu Ser Phe Met Arg Thr Asp Asp Ile Gly Thr Ala

195 200 205

Ser Gly Trp Gly Leu Thr Gln Arg Gly Phe Leu Ala Arg Asn Leu Met

210 215 220

Tyr Val Asp Ile Pro Ile Val Asp His Gln Lys Cys Thr Ala Ala Tyr

225 230 235 240

Glu Lys Pro Pro Tyr Pro Arg Gly Ser Val Thr Ala Asn Met Leu Cys

245 250 255

Ala Gly Leu Glu Ser Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly

260 265 270

Gly Ala Leu Val Phe Leu Asp Ser Glu Thr Glu Arg Trp Phe Val Gly

275 280 285

Gly Ile Val Ser Trp Gly Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr

290 295 300

Gly Val Tyr Thr Lys Val Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile

305 310 315 320

Ile Ser Asp Phe

<210> 15

<211> 82

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 15

Ile Val Asp Cys Gly Pro Pro Asp Asp Leu Pro Ser Gly Arg Val Glu

1 5 10 15

Tyr Ile Thr Gly Pro Gly Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr

20 25 30

Ser Cys Glu Glu Thr Phe Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr

35 40 45

Val Cys Glu Ala Asp Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser

50 55 60

Leu Pro Val Cys Glu Pro Val Cys Gly Leu Ser Ala Arg Thr Thr Gly

65 70 75 80

Gly Arg

<210> 16

<211> 242

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 16

Ile Tyr Gly Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val

1 5 10 15

Leu Ile Leu Gly Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr Asp Asn

20 25 30

Trp Val Leu Thr Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala

35 40 45

Ser Ala Leu Asp Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His

50 55 60

Tyr Thr Gln Ala Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr

65 70 75 80

His Asp Ala Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn

85 90 95

Lys Val Val Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys

100 105 110

Glu Ala Glu Ser Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly

115 120 125

Trp Gly Leu Thr Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val

130 135 140

Asp Ile Pro Ile Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys

145 150 155 160

Pro Pro Tyr Pro Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly

165 170 175

Leu Glu Ser Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala

180 185 190

Leu Val Phe Leu Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile

195 200 205

Val Ser Trp Gly Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val

210 215 220

Tyr Thr Lys Val Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser

225 230 235 240

Asp Phe

<210> 17

<211> 16

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 17

Gly Lys Asp Ser Cys Arg Gly Asp Ala Gly Gly Ala Leu Val Phe Leu

1 5 10 15

<210> 18

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 18

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Gly

20 25 30

Lys Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr Arg Thr Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Arg Ala Gly Gly Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 19

<211> 354

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<400> 19

caggtcacct tgaaggagtc tggtcctgtg ctggtgaaac ccacagagac cctcacgctg 60

acctgcaccg tctctgggtt ctcactcagc aggggtaaaa tgggtgtgag ctggatccgt 120

cagcccccag ggaaggccct ggagtggctt gcacacattt tttcgagtga cgaaaaatcc 180

tacaggacat cgctgaagag caggctcacc atctccaagg acacctccaa aaaccaggtg 240

gtccttacaa tgaccaacat ggaccctgtg gacacagcca cgtattactg tgcacggata 300

cgacgtggag gaattgacta ctggggccag ggaaccctgg tcactgtctc ctca 354

<210> 20

<211> 118

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 20

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Gly

20 25 30

Lys Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr Arg Thr Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Arg Arg Gly Gly Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 21

<211> 121

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 21

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Thr

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Val Pro Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 22

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 22

Gln Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Phe Ala

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly His Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Gly Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val

85 90 95

Phe Gly Thr Gly Thr Lys Val Thr Val Leu Ala

100 105

<210> 23

<211> 318

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<220>

<221>Further feature

<222> (246)..(246)

<223>N is a, c, g, or t

<400> 23

cagccagtgc tgactcagcc cccctcactg tccgtgtccc caggacagac agccagcatc 60

acctgctctg gagagaaatt gggggataaa tatgcttact ggtatcagca gaagccaggc 120

cagtcccctg tgttggtcat gtatcaagat aaacagcggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240

gatgangctg actattactg tcaggcgtgg gacagcagca ctgcggtatt cggcggaggg 300

accaagctga ccgtccta 318

<210> 24

<211> 106

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 24

Gln Pro Val Leu Thr Gln Pro Pro Ser Leu Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Glu Lys Leu Gly Asp Lys Tyr Ala

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Met Tyr

35 40 45

Gln Asp Lys Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 25

<211> 120

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 25

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Val Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Thr Thr Asp His

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly

100 105 110

Ser Glu Gln Lys Leu Ile Ser Glu

115 120

<210> 26

<211> 324

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<400> 26

tcctatgagc tgatacagcc accctcggtg tcagtggccc caggacagac ggccaccatt 60

acctgtgcgg gagacaacct tgggaagaaa cgtgtgcact ggtaccagca gaggccaggc 120

caggcccctg tgttggtcat ctatgatgat agcgaccggc cctcagggat ccctgaccga 180

ttctctgcct ccaactctgg gaacacggcc accctgacca tcactagggg cgaagccggg 240

gatgaggccg actattattg tcaggtgtgg gacattgcta ctgatcatgt ggtcttcggc 300

ggagggacca agctcaccgt ccta 324

<210> 27

<211> 120

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 27

Ser Tyr Glu Leu Ile Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Thr Ile Thr Cys Ala Gly Asp Asn Leu Gly Lys Lys Arg Val

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Ala Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Thr Arg Gly Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ile Ala Thr Asp His

85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly

100 105 110

Ser Glu Gln Lys Leu Ile Ser Glu

115 120

<210> 28

<211> 5

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 28

Arg Gly Lys Met Gly

1 5

<210> 29

<211> 5

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 29

Ser Thr Ser Ala Ala

1 5

<210> 30

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 30

Gly Phe Ser Leu Ser Arg Gly

1 5

<210> 31

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 31

Gly Asp Ser Val Ser Ser Thr

1 5

<210> 32

<211> 16

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 32

Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr Arg Thr Ser Leu

1 5 10 15

<210> 33

<211> 16

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 33

Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val

1 5 10 15

<210> 34

<211> 5

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 34

His Ile Phe Ser Ser

1 5

<210> 35

<211> 5

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 35

Arg Thr Tyr Tyr Arg

1 5

<210> 36

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 36

Tyr Tyr Cys Ala Arg Ile Arg Ala

1 5

<210> 37

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 37

Tyr Tyr Cys Ala Arg Ile Arg Arg

1 5

<210> 38

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 38

Ala Val Tyr Tyr Cys Ala Arg Asp

1 5

<210> 39

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 39

Tyr Tyr Cys Ala Arg Ile Arg

1 5

<210> 40

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 40

Ala Val Tyr Tyr Cys Ala Arg

1 5

<210> 41

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 41

Gly Asp Lys Leu Gly Asp Lys Phe Ala Tyr Trp

1 5 10

<210> 42

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 42

Gly Glu Lys Leu Gly Asp Lys Tyr Ala Tyr Trp

1 5 10

<210> 43

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 43

Gly Asn Asn Ile Gly Ser Lys Asn Val His Trp

1 5 10

<210> 44

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 44

Gly Asp Asn Leu Gly Lys Lys Arg Val His Trp

1 5 10

<210> 45

<211> 16

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 45

Ala Gly Asp Asn Leu Gly Lys Lys Arg Val His Trp Tyr Gln Gln Arg

1 5 10 15

<210> 46

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 46

Asp Asn Lys Arg Pro Ser Gly

1 5

<210> 47

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 47

Asp Lys Gln Arg Pro Ser Gly

1 5

<210> 48

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 48

Asp Lys Gln Arg Pro Ser Gly Ile Pro Glu Arg

1 5 10

<210> 49

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 49

Asp Ser Asp Arg Pro Ser Gly

1 5

<210> 50

<211> 14

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 50

Asp Ser Asp Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Ala

1 5 10

<210> 51

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 51

Ala Trp Asp Ser Ser Thr Ala Val Phe

1 5

<210> 52

<211> 16

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 52

Ala Trp Asp Ser Ser Thr Ala Val Phe Gly Gly Gly Thr Lys Leu Thr

1 5 10 15

<210> 53

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 53

Val Trp Asp Thr Thr Thr Asp His Val

1 5

<210> 54

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 54

Val Trp Asp Ile Ala Thr Asp His Val

1 5

<210> 55

<211> 268

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 55

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Gly

20 25 30

Lys Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr Arg Thr Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Arg Ala Gly Gly Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Lys Leu Ser Gly Ser Ala Ser Ala Pro Lys

115 120 125

Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Gln Pro Val Leu Thr

130 135 140

Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr Ala Ser Ile Thr

145 150 155 160

Cys Ser Gly Asp Lys Leu Gly Asp Lys Phe Ala Tyr Trp Tyr Gln Gln

165 170 175

Lys Pro Gly His Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys Arg

180 185 190

Pro Ser Gly Ile Pro Gly Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr

195 200 205

Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr

210 215 220

Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val Phe Gly Thr Gly Thr

225 230 235 240

Lys Val Thr Val Leu Ala Ala Ala Gly Ser Glu Gln Lys Leu Ile Ser

245 250 255

Glu Glu Asp Leu Asn Ser His His His His His His

260 265

<210> 56

<211> 271

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 56

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Ala

20 25 30

Arg Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Phe Ser Asn Asp Glu Lys Ser Tyr Ser Thr Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Met Ala Ile Leu Thr Ala Gly Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Lys Leu Ser Gly Ser Ala Ser

115 120 125

Ala Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Ser Tyr

130 135 140

Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ser Pro Gly Gln Thr Ala

145 150 155 160

Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Val Ser Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Arg Ser Pro Val Leu Val Ile Tyr Arg Asp

180 185 190

Thr Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Tyr

195 200 205

Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Val Asp Glu

210 215 220

Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Gly Ser Gly Val Phe Gly

225 230 235 240

Gly Gly Thr Lys Val Thr Val Leu Ala Ala Ala Gly Ser Glu Gln Lys

245 250 255

Leu Ile Ser Glu Glu Asp Leu Asn Ser His His His His His His

260 265 270

<210> 57

<211> 272

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 57

Gln Ile Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Ala

20 25 30

Arg Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Phe Ser Asn Asp Glu Lys Ser Tyr Ser Thr Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Met Arg Tyr Ser Ser Ser Leu Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Arg Val Thr Val Ser Ser Lys Leu Ser Gly Ser Ala

115 120 125

Ser Ala Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Ser

130 135 140

Tyr Glu Leu Ile Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr

145 150 155 160

Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Val Ser

165 170 175

Trp Tyr Gln Gln Lys Pro Gly Arg Ser Pro Val Leu Val Ile Tyr Arg

180 185 190

Asp Thr Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn

195 200 205

Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp

210 215 220

Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Val Val Phe

225 230 235 240

Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly Ser Glu Gln

245 250 255

Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser His His His His His His

260 265 270

<210> 58

<211> 273

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 58

Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Lys

20 25 30

Arg Ala Ala Trp Asp Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Phe Asn Asp Tyr Ala

50 55 60

Ile Ser Val Lys Ser Arg Ile Thr Ile Asn Ala Asp Thr Ser Arg Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Val Lys Ser Asn Ser Gly Thr Gly Ala Phe Asp Ser Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Lys Leu Ser Gly Ser Ala

115 120 125

Ser Ala Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Gln

130 135 140

Pro Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr

145 150 155 160

Ala Arg Ile Thr Cys Ser Arg Asp Lys Leu Gly Asp Lys Tyr Val Ser

165 170 175

Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Val Val Met Tyr Lys

180 185 190

Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn

195 200 205

Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp

210 215 220

Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Gly Val

225 230 235 240

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly Ser Glu

245 250 255

Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser His His His His His

260 265 270

His

<210> 59

<211> 273

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 59

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Thr

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Val Pro Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser Lys Leu Ser Gly Ser Ala Ser

115 120 125

Ala Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Ser Tyr

130 135 140

Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala

145 150 155 160

Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val His Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp

180 185 190

Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser

195 200 205

Gly Asn Thr Ala Thr Leu Thr Val Ser Arg Val Glu Ala Gly Asp Glu

210 215 220

Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Thr Thr Asp His Val Val

225 230 235 240

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly Ser Glu

245 250 255

Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser His His His His His

260 265 270

His

<210> 60

<211> 273

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 60

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Val Pro Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser Lys Leu Ser Gly Ser Ala Ser

115 120 125

Ala Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Ser Tyr

130 135 140

Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala

145 150 155 160

Arg Val Thr Cys Gly Gly Asn Asn Ile Arg Ser Lys Asn Val His Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp

180 185 190

Thr Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser

195 200 205

Gly Asn Thr Ala Thr Leu Thr Val Ser Arg Val Glu Ala Gly Asp Glu

210 215 220

Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Thr Thr Asp His Val Val

225 230 235 240

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly Ser Glu

245 250 255

Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser His His His His His

260 265 270

His

<210> 61

<211> 273

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 61

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Val Pro Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser Lys Leu Ser Gly Ser Ala Ser

115 120 125

Ala Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Ser Tyr

130 135 140

Glu Leu Thr Gln Leu Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala

145 150 155 160

Arg Val Thr Cys Gly Gly Asn Asn Ile Arg Ser Lys Asn Val His Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp Asp

180 185 190

Thr Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser

195 200 205

Gly Asn Thr Ala Thr Leu Thr Val Ser Arg Val Glu Ala Gly Asp Glu

210 215 220

Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Thr Thr Asp His Val Val

225 230 235 240

Phe Gly Gly Gly Thr Lys Val Thr Val Leu Ala Ala Ala Gly Ser Glu

245 250 255

Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser His His His His His

260 265 270

His

<210> 62

<211> 984

<212> DNA

<213>Homo sapiens

<400> 62

gcctccacca agggcccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 60

agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240

tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300

aaatatggtc ccccatgccc atcatgccca gcacctgagt tcctgggggg accatcagtc 360

ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420

tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600

tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660

gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720

aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840

gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 900

aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960

ctctccctgt ctctcgggaa atga 984

<210> 63

<211> 327

<212> PRT

<213>Homo sapiens

<400> 63

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 64

<211> 984

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<400> 64

gcctccacca agggcccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 60

agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240

tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300

aaatatggtc ccccatgccc accatgccca gcacctgagt tcctgggggg accatcagtc 360

ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420

tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600

tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660

gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720

aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840

gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 900

aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960

ctctccctgt ctctcgggaa atga 984

<210> 65

<211> 327

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 65

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 66

<211> 262

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 66

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Thr

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Val Pro Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser Lys Leu Ser Gly Ser Ala Ser

115 120 125

Ala Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Ser Tyr

130 135 140

Glu Leu Ile Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala

145 150 155 160

Thr Ile Thr Cys Ala Gly Asp Asn Leu Gly Lys Lys Arg Val His Trp

165 170 175

Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Asp Asp

180 185 190

Ser Asp Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Ala Ser Asn Ser

195 200 205

Gly Asn Thr Ala Thr Leu Thr Ile Thr Arg Gly Glu Ala Gly Asp Glu

210 215 220

Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ile Ala Thr Asp His Val Val

225 230 235 240

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly Ser Glu

245 250 255

Gln Lys Leu Ile Ser Glu

260

<210> 67

<211> 245

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 67

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Gly

20 25 30

Lys Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr Arg Thr Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Arg Ala Gly Gly Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Lys Leu Ser Gly Ser Ala Ser Ala Pro Lys

115 120 125

Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Gln Pro Val Leu Thr

130 135 140

Gln Pro Pro Ser Leu Ser Val Ser Pro Gly Gln Thr Ala Ser Ile Thr

145 150 155 160

Cys Ser Gly Glu Lys Leu Gly Asp Lys Tyr Ala Tyr Trp Tyr Gln Gln

165 170 175

Lys Pro Gly Gln Ser Pro Val Leu Val Met Tyr Gln Asp Lys Gln Arg

180 185 190

Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr

195 200 205

Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr

210 215 220

Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val Phe Gly Gly Gly Thr

225 230 235 240

Lys Leu Thr Val Leu

245

<210> 68

<211> 245

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 68

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Gly

20 25 30

Lys Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr Arg Thr Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Arg Arg Gly Gly Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Lys Leu Ser Gly Ser Ala Ser Ala Pro Lys

115 120 125

Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Gln Pro Val Leu Thr

130 135 140

Gln Pro Pro Ser Leu Ser Val Ser Pro Gly Gln Thr Ala Ser Ile Thr

145 150 155 160

Cys Ser Gly Glu Lys Leu Gly Asp Lys Tyr Ala Tyr Trp Tyr Gln Gln

165 170 175

Lys Pro Gly Gln Ser Pro Val Leu Val Met Tyr Gln Asp Lys Gln Arg

180 185 190

Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr

195 200 205

Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr

210 215 220

Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val Phe Gly Gly Gly Thr

225 230 235 240

Lys Leu Thr Val Leu

245

<210> 69

<211> 981

<212> DNA

<213>Homo sapiens

<400> 69

gcctccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 60

agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 240

tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 300

aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 360

ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 420

gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 480

gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 540

gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga gtacaagtgc 600

aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 660

cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 720

caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtagagtgg 780

gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 840

ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 900

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 960

tccctgtctc ccgggaaatg a 981

<210> 70

<211> 326

<212> PRT

<213>Homo sapiens

<400> 70

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 71

<211> 702

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<400> 71

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggcctcc 60

tatgagctga tacagccacc ctcggtgtca gtggccccag gacagacggc caccattacc 120

tgtgcgggag acaaccttgg gaagaaacgt gtgcactggt accagcagag gccaggccag 180

gcccctgtgt tggtcatcta tgatgatagc gaccggccct cagggatccc tgaccgattc 240

tctgcctcca actctgggaa cacggccacc ctgaccatca ctaggggcga agccggggat 300

gaggccgact attattgtca ggtgtgggac attgctactg atcatgtggt cttcggcgga 360

gggaccaagc tcaccgtcct aggccagcct aaggcggcgc cctcggtcac cctgttcccg 420

ccctcctctg aggagcttca agccaacaag gccacactgg tgtgtctcat aagtgacttc 480

tacccgggag ccgtgacagt ggcctggaag gcagatagca gccccgtcaa ggcgggagtg 540

gagaccacca caccctccaa acaaagcaac aacaagtacg cggccagcag ctatctgagc 600

ctgacgcctg agcagtggaa gtcccacaga agctacagct gccaggtcac gcatgaaggg 660

agcaccgtgg agaagacagt ggcccctaca gaatgttcat ag 702

<210> 72

<211> 233

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 72

Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala

1 5 10 15

Thr Gln Ala Ser Tyr Glu Leu Ile Gln Pro Pro Ser Val Ser Val Ala

20 25 30

Pro Gly Gln Thr Ala Thr Ile Thr Cys Ala Gly Asp Asn Leu Gly Lys

35 40 45

Lys Arg Val His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu

50 55 60

Val Ile Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Asp Arg Phe

65 70 75 80

Ser Ala Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Thr Arg Gly

85 90 95

Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ile Ala

100 105 110

Thr Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

115 120 125

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

130 135 140

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

145 150 155 160

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

165 170 175

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

180 185 190

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

195 200 205

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

210 215 220

Lys Thr Val Ala Pro Thr Glu Cys Ser

225 230

<210> 73

<211> 1401

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<400> 73

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggcccag 60

gtacagctgc agcagtcagg tccaggactg gtgaagccct cgcagaccct ctcactcacc 120

tgtgccatct ccggggacag tgtctctagc accagtgctg cttggaactg gatcaggcag 180

tccccatcga gaggccttga gtggctggga aggacatact acaggtccaa gtggtataat 240

gattatgcag tatctgtgaa aagtcgaata accatcaacc cagacacatc caagaaccag 300

ttctccctgc agctgaactc tgtgactccc gaggacacgg ctgtgtatta ctgtgcaaga 360

gatcctttcg gggtaccttt tgatatctgg ggccaaggga caatggtcac cgtctcttca 420

gcctccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 480

agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540

tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca 600

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 660

tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 720

aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 780

ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 840

gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 900

gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 960

gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga gtacaagtgc 1020

aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 1080

cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140

caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtagagtgg 1200

gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 1260

ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1320

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1380

tccctgtctc ccgggaaatg a 1401

<210> 74

<211> 466

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 74

Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala

1 5 10 15

Thr Gln Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys

20 25 30

Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val

35 40 45

Ser Ser Thr Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg

50 55 60

Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn

65 70 75 80

Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr

85 90 95

Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Val Pro Phe Asp

115 120 125

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

145 150 155 160

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn

210 215 220

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg

225 230 235 240

Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210> 75

<211> 1404

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<400> 75

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggcccag 60

gtacagctgc agcagtcagg tccaggactg gtgaagccct cgcagaccct ctcactcacc 120

tgtgccatct ccggggacag tgtctctagc accagtgctg cttggaactg gatcaggcag 180

tccccatcga gaggccttga gtggctggga aggacatact acaggtccaa gtggtataat 240

gattatgcag tatctgtgaa aagtcgaata accatcaacc cagacacatc caagaaccag 300

ttctccctgc agctgaactc tgtgactccc gaggacacgg ctgtgtatta ctgtgcaaga 360

gatcctttcg gggtaccttt tgatatctgg ggccaaggga caatggtcac cgtctcttca 420

gcctccacca agggcccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 480

agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 660

tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 720

aaatatggtc ccccatgccc atcatgccca gcacctgagt tcctgggggg accatcagtc 780

ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 840

tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 1320

aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 1380

ctctccctgt ctctcgggaa atga 1404

<210> 76

<211> 467

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 76

Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala

1 5 10 15

Thr Gln Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys

20 25 30

Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val

35 40 45

Ser Ser Thr Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg

50 55 60

Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn

65 70 75 80

Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr

85 90 95

Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Val Pro Phe Asp

115 120 125

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

145 150 155 160

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

210 215 220

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

225 230 235 240

Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

275 280 285

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Leu Gly Lys

465

<210> 77

<211> 1404

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<400> 77

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggcccag 60

gtacagctgc agcagtcagg tccaggactg gtgaagccct cgcagaccct ctcactcacc 120

tgtgccatct ccggggacag tgtctctagc accagtgctg cttggaactg gatcaggcag 180

tccccatcga gaggccttga gtggctggga aggacatact acaggtccaa gtggtataat 240

gattatgcag tatctgtgaa aagtcgaata accatcaacc cagacacatc caagaaccag 300

ttctccctgc agctgaactc tgtgactccc gaggacacgg ctgtgtatta ctgtgcaaga 360

gatcctttcg gggtaccttt tgatatctgg ggccaaggga caatggtcac cgtctcttca 420

gcctccacca agggcccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 480

agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 660

tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 720

aaatatggtc ccccatgccc accatgccca gcacctgagt tcctgggggg accatcagtc 780

ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 840

tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 1320

aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 1380

ctctccctgt ctctcgggaa atga 1404

<210> 78

<211> 467

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 78

Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala

1 5 10 15

Thr Gln Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys

20 25 30

Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val

35 40 45

Ser Ser Thr Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg

50 55 60

Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn

65 70 75 80

Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr

85 90 95

Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Val Pro Phe Asp

115 120 125

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

145 150 155 160

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

210 215 220

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

225 230 235 240

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

275 280 285

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Leu Gly Lys

465

<210> 79

<211> 696

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<220>

<221>Further feature

<222> (303)..(303)

<223>N is a, c, g, or t

<400> 79

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggcccag 60

ccagtgctga ctcagccccc ctcactgtcc gtgtccccag gacagacagc cagcatcacc 120

tgctctggag agaaattggg ggataaatat gcttactggt atcagcagaa gccaggccag 180

tcccctgtgt tggtcatgta tcaagataaa cagcggccct cagggatccc tgagcgattc 240

tctggctcca actctgggaa cacagccact ctgaccatca gcgggaccca ggctatggat 300

gangctgact attactgtca ggcgtgggac agcagcactg cggtattcgg cggagggacc 360

aagctgaccg tcctaggcca gcctaaggcg gcgccctcgg tcaccctgtt cccgccctcc 420

tctgaggagc ttcaagccaa caaggccaca ctggtgtgtc tcataagtga cttctacccg 480

ggagccgtga cagtggcctg gaaggcagat agcagccccg tcaaggcggg agtggagacc 540

accacaccct ccaaacaaag caacaacaag tacgcggcca gcagctatct gagcctgacg 600

cctgagcagt ggaagtccca cagaagctac agctgccagg tcacgcatga agggagcacc 660

gtggagaaga cagtggcccc tacagaatgt tcatag 696

<210> 80

<211> 231

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 80

Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala

1 5 10 15

Thr Gln Ala Gln Pro Val Leu Thr Gln Pro Pro Ser Leu Ser Val Ser

20 25 30

Pro Gly Gln Thr Ala Ser Ile Thr Cys Ser Gly Glu Lys Leu Gly Asp

35 40 45

Lys Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu

50 55 60

Val Met Tyr Gln Asp Lys Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe

65 70 75 80

Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr

85 90 95

Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser

100 105 110

Thr Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

115 120 125

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

130 135 140

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

145 150 155 160

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

165 170 175

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

180 185 190

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

195 200 205

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

210 215 220

Val Ala Pro Thr Glu Cys Ser

225 230

<210> 81

<211> 1392

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<400> 81

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggcccag 60

gtcaccttga aggagtctgg tcctgtgctg gtgaaaccca cagagaccct cacgctgacc 120

tgcaccgtct ctgggttctc actcagcagg ggtaaaatgg gtgtgagctg gatccgtcag 180

cccccaggga aggccctgga gtggcttgca cacatttttt cgagtgacga aaaatcctac 240

aggacatcgc tgaagagcag gctcaccatc tccaaggaca cctccaaaaa ccaggtggtc 300

cttacaatga ccaacatgga ccctgtggac acagccacgt attactgtgc acggatacga 360

cgtggaggaa ttgactactg gggccaggga accctggtca ctgtctcctc agcctccacc 420

aagggcccat cggtcttccc cctggcgccc tgctccagga gcacctccga gagcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgctctga ccagcggcgt gcacaccttc ccagctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcaacttcg gcacccagac ctacacctgc 660

aacgtagatc acaagcccag caacaccaag gtggacaaga cagttgagcg caaatgttgt 720

gtcgagtgcc caccgtgccc agcaccacct gtggcaggac cgtcagtctt cctcttcccc 780

ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacgtg cgtggtggtg 840

gacgtgagcc acgaagaccc cgaggtccag ttcaactggt acgtggacgg cgtggaggtg 900

cataatgcca agacaaagcc acgggaggag cagttcaaca gcacgttccg tgtggtcagc 960

gtcctcaccg ttgtgcacca ggactggctg aacggcaagg agtacaagtg caaggtctcc 1020

aacaaaggcc tcccagcccc catcgagaaa accatctcca aaaccaaagg gcagccccga 1080

gaaccacagg tgtacaccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc 1140

ctgacctgcc tggtcaaagg cttctacccc agcgacatcg ccgtagagtg ggagagcaat 1200

gggcagccgg agaacaacta caagaccaca cctcccatgc tggactccga cggctccttc 1260

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1320

tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1380

cccgggaaat ga 1392

<210> 82

<211> 463

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 82

Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala

1 5 10 15

Thr Gln Ala Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys

20 25 30

Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Ser Arg Gly Lys Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Ala Leu Glu Trp Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr

65 70 75 80

Arg Thr Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys

85 90 95

Asn Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala

100 105 110

Thr Tyr Tyr Cys Ala Arg Ile Arg Arg Gly Gly Ile Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys

225 230 235 240

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

245 250 255

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

260 265 270

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

275 280 285

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

290 295 300

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

305 310 315 320

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

325 330 335

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

340 345 350

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

355 360 365

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

370 375 380

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

385 390 395 400

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

405 410 415

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

420 425 430

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

435 440 445

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455 460

<210> 83

<211> 1395

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<400> 83

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggcccag 60

gtcaccttga aggagtctgg tcctgtgctg gtgaaaccca cagagaccct cacgctgacc 120

tgcaccgtct ctgggttctc actcagcagg ggtaaaatgg gtgtgagctg gatccgtcag 180

cccccaggga aggccctgga gtggcttgca cacatttttt cgagtgacga aaaatcctac 240

aggacatcgc tgaagagcag gctcaccatc tccaaggaca cctccaaaaa ccaggtggtc 300

cttacaatga ccaacatgga ccctgtggac acagccacgt attactgtgc acggatacga 360

cgtggaggaa ttgactactg gggccaggga accctggtca ctgtctcctc agcctccacc 420

aagggcccat ccgtcttccc cctggcgccc tgctccagga gcacctccga gagcacagcc 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacgaagac ctacacctgc 660

aacgtagatc acaagcccag caacaccaag gtggacaaga gagttgagtc caaatatggt 720

cccccatgcc catcatgccc agcacctgag ttcctggggg gaccatcagt cttcctgttc 780

cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 840

gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 900

gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 960

agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 1020

tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 1080

cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 1140

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 1200

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1260

ttcttcctct acagcaggct aaccgtggac aagagcaggt ggcaggaggg gaatgtcttc 1320

tcatgctccg tgatgcatga ggctctgcac aaccactaca cacagaagag cctctccctg 1380

tctctcggga aatga 1395

<210> 84

<211> 464

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 84

Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala

1 5 10 15

Thr Gln Ala Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys

20 25 30

Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Ser Arg Gly Lys Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Ala Leu Glu Trp Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr

65 70 75 80

Arg Thr Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys

85 90 95

Asn Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala

100 105 110

Thr Tyr Tyr Cys Ala Arg Ile Arg Arg Gly Gly Ile Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

225 230 235 240

Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

275 280 285

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

450 455 460

<210> 85

<211> 1395

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<400> 85

atgatgtcct ttgtctctct gctcctggtt ggcatcctat tccatgccac ccaggcccag 60

gtcaccttga aggagtctgg tcctgtgctg gtgaaaccca cagagaccct cacgctgacc 120

tgcaccgtct ctgggttctc actcagcagg ggtaaaatgg gtgtgagctg gatccgtcag 180

cccccaggga aggccctgga gtggcttgca cacatttttt cgagtgacga aaaatcctac 240

aggacatcgc tgaagagcag gctcaccatc tccaaggaca cctccaaaaa ccaggtggtc 300

cttacaatga ccaacatgga ccctgtggac acagccacgt attactgtgc acggatacga 360

cgtggaggaa ttgactactg gggccaggga accctggtca ctgtctcctc agcctccacc 420

aagggcccat ccgtcttccc cctggcgccc tgctccagga gcacctccga gagcacagcc 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacgaagac ctacacctgc 660

aacgtagatc acaagcccag caacaccaag gtggacaaga gagttgagtc caaatatggt 720

cccccatgcc caccatgccc agcacctgag ttcctggggg gaccatcagt cttcctgttc 780

cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 840

gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 900

gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 960

agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 1020

tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 1080

cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 1140

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 1200

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1260

ttcttcctct acagcaggct aaccgtggac aagagcaggt ggcaggaggg gaatgtcttc 1320

tcatgctccg tgatgcatga ggctctgcac aaccactaca cacagaagag cctctccctg 1380

tctctcggga aatga 1395

<210> 86

<211> 464

<212> PRT

<213>Artificial sequence

<220>

<223>Synthesis

<400> 86

Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala

1 5 10 15

Thr Gln Ala Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys

20 25 30

Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Ser Arg Gly Lys Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Ala Leu Glu Trp Leu Ala His Ile Phe Ser Ser Asp Glu Lys Ser Tyr

65 70 75 80

Arg Thr Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys

85 90 95

Asn Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala

100 105 110

Thr Tyr Tyr Cys Ala Arg Ile Arg Arg Gly Gly Ile Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

225 230 235 240

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

275 280 285

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

450 455 460

<210> 87

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<400> 87

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaccagtg ctgcttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattatg cagtatctgt gaaaagtcga ataaccatca acccagacac atccaagaac 240

cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300

agagatcctt tcggggtacc ttttgatatc tggggccaag ggacaatggt caccgtctct 360

tcaaagcttt cagggagtgc atccgcccca aaacttgaag aaggtgaatt ttcagaagca 420

cgcgtatcct atgagctgat acagccaccc tcggtgtcag tggccccagg acagacggcc 480

accattacct gtgcgggaga caaccttggg aagaaacgtg tgcactggta ccagcagagg 540

ccaggccagg cccctgtgtt ggtcatctat gatgatagcg accggccctc agggatccct 600

gaccgattct ctgcctccaa ctctgggaac acggccaccc tgaccatcac taggggcgaa 660

gccggggatg aggccgacta ttattgtcag gtgtgggaca ttgctactga tcatgtggtc 720

ttcggcggag ggaccaagct caccgtccta 750

<210> 88

<211> 735

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<220>

<221>Further feature

<222> (663)..(663)

<223>N is a, c, g, or t

<400> 88

caggtcacct tgaaggagtc tggtcctgtg ctggtgaaac ccacagagac cctcacgctg 60

acctgcaccg tctctgggtt ctcactcagc aggggtaaaa tgggtgtgag ctggatccgt 120

cagcccccag ggaaggccct ggagtggctt gcacacattt tttcgagtga cgaaaaatcc 180

tacaggacat cgctgaagag caggctcacc atctccaagg acacctccaa aaaccaggtg 240

gtccttacaa tgaccaacat ggaccctgtg gacacagcca cgtattactg tgcacggata 300

cgagcgggag gaattgacta ctggggccag ggaaccctgg tcactgtctc ctcaaagctt 360

tcagggagtg catccgcccc aaaacttgaa gaaggtgaat tttcagaagc acgcgtacag 420

ccagtgctga ctcagccccc ctcactgtcc gtgtccccag gacagacagc cagcatcacc 480

tgctctggag agaaattggg ggataaatat gcttactggt atcagcagaa gccaggccag 540

tcccctgtgt tggtcatgta tcaagataaa cagcggccct cagggatccc tgagcgattc 600

tctggctcca actctgggaa cacagccact ctgaccatca gcgggaccca ggctatggat 660

gangctgact attactgtca ggcgtgggac agcagcactg cggtattcgg cggagggacc 720

aagctgaccg tccta 735

<210> 89

<211> 735

<212> DNA

<213>Artificial sequence

<220>

<223>Synthesis

<220>

<221>Further feature

<222> (663)..(663)

<223>N is a, c, g, or t

<400> 89

caggtcacct tgaaggagtc tggtcctgtg ctggtgaaac ccacagagac cctcacgctg 60

acctgcaccg tctctgggtt ctcactcagc aggggtaaaa tgggtgtgag ctggatccgt 120

cagcccccag ggaaggccct ggagtggctt gcacacattt tttcgagtga cgaaaaatcc 180

tacaggacat cgctgaagag caggctcacc atctccaagg acacctccaa aaaccaggtg 240

gtccttacaa tgaccaacat ggaccctgtg gacacagcca cgtattactg tgcacggata 300

cgacgtggag gaattgacta ctggggccag ggaaccctgg tcactgtctc ctcaaagctt 360

tcagggagtg catccgcccc aaaacttgaa gaaggtgaat tttcagaagc acgcgtacag 420

ccagtgctga ctcagccccc ctcactgtcc gtgtccccag gacagacagc cagcatcacc 480

tgctctggag agaaattggg ggataaatat gcttactggt atcagcagaa gccaggccag 540

tcccctgtgt tggtcatgta tcaagataaa cagcggccct cagggatccc tgagcgattc 600

tctggctcca actctgggaa cacagccact ctgaccatca gcgggaccca ggctatggat 660

gangctgact attactgtca ggcgtgggac agcagcactg cggtattcgg cggagggacc 720

aagctgaccg tccta 735

<210> 90

<211> 8

<212> PRT

<213>Artificial sequence

<220>

<223> consensus CDRH3 of 17D20m and d3521N11

<220>

<221> VARIANT

<222> (8)..(8)

<223> wherein Xaa at position 8 is Ala or Arg

<400> 90

Tyr Tyr Cys Ala Arg Ile Arg Xaa

1 5

<210> 91

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223> consensus of CDRL1 of 17D20m and d3521N11

<220>

<221> VARIANT

<222> (2)..(2)

<223> wherein Xaa at position 2 is Asp or Glu

<220>

<221> VARIANT

<222> (8)..(8)

<223> wherein Xaa at position 8 is Phe or Tyr

<400> 91

Gly Xaa Lys Leu Gly Asp Lys Xaa Ala Tyr Trp

1 5 10

<210> 92

<211> 11

<212> PRT

<213>Artificial sequence

<220>

<223>17N16m and d17N9 shared CDRL1

<220>

<221>Variant

<222> (2)..(2)

<223>The Xaa of wherein position 2 is Asn or Asp

<220>

<221>Variant

<222> (4)..(4)

<223>The Xaa of wherein position 4 is Ile or Leu

<220>

<221> VARIANT

<222> (6)..(6)

<223> wherein Xaa at position 6 is Ser or Lys

<220>

<221>Variant

<222> (8)..(8)

<223>The Xaa of wherein position 8 is Asn or Arg

<400> 92

Gly Xaa Asn Xaa Gly Xaa Lys Xaa Val His Trp

1 5 10

<210> 93

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223>17D20m and d3521N11 shared CDRL2

<220>

<221>Variant

<222> (2)..(2)

<223>The Xaa of wherein position 2 is Asn or Lys or Ser

<220>

<221>Variant

<222> (3)..(3)

<223>The Xaa of wherein position 3 is Lys or Gln or Asp

<400> 93

Asp Xaa Xaa Arg Pro Ser Gly

1 5

<210> 94

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223>17N16m and d17N9 shared CDRL3

<220>

<221>Variant

<222> (4)..(4)

<223>The Xaa of wherein position 4 is Thr or Ile

<220>

<221>Variant

<222> (5)..(5)

<223>The Xaa of wherein position 5 is Thr or Ala

<400> 94

Val Trp Asp Xaa Xaa Thr Asp His Val

1 5

Claims (16)

1. the human monoclonal antibodies or its antigen-binding fragment of separation, it combines people MASP-2 and suppresses MASP-2 dependences benefit Body is activated, wherein the antibody or its antigen-binding fragment include：

i）Weight chain variable district, it includes 3 complementary determining regions CDR-H1, CDR-H2 and CDR-H3；With

ii）Light chain variable district, it includes 3 CDR, i.e. CDR-L1, CDR-L2 and CDR-L3；Wherein

CDR-H1 is by SEQ ID NO：Amino acid sequence composition shown in 29；

CDR-H2 is by SEQ ID NO：Amino acid sequence composition shown in 33；

CDR-H3 is by SEQ ID NO：Amino acid sequence composition shown in 38；

CDR-L1 is by SEQ ID NO：Amino acid sequence composition shown in 92, wherein the X of the 2nd is N or D, and wherein the 4th X is I or L, and the X of wherein the 6th is S or K, and the X of wherein the 8th is N or R；

CDR-L2 is by SEQ ID NO：Amino acid sequence composition shown in 49；With

CDR-L3 is by SEQ ID NO：Amino acid sequence composition shown in 94, wherein the X of the 4th is T or I, and wherein the 5th X is T or A；Wherein the antibody of the separation or its antigen-binding fragment are combined with people MASP-2 and suppress MASP-2 dependences benefit Body is activated.

2. the antibody of the separation of claim 1, wherein CDR-L1 are by SEQ ID NO：Amino acid sequence composition shown in 92, wherein The X of the 2nd is D, wherein the X of the 4th is L, wherein the X of the 6th is K, and the X of wherein the 8th is R, and wherein CDR-L2 is by SEQ ID NO：Amino acid sequence composition shown in 49, and wherein CDR-L3 is by SEQ ID NO：Amino acid sequence composition shown in 94, The X of wherein the 4th is I, and the X of wherein the 5th is A.

3. the antibody of the separation of claim 1, wherein CDR-L1 are by SEQ ID NO：Amino acid sequence composition shown in 92, wherein The X of the 2nd is N, wherein the X of the 4th is I, wherein the X of the 6th is S, wherein the X of the 8th is N, wherein CDR-L2 is by SEQ ID NO：Amino acid sequence composition shown in 49, and wherein CDR-L3 is by SEQ ID NO：Amino acid sequence composition shown in 94, The X of wherein the 4th is that the X at T and the wherein the 5th is T.

4. antibody according to claim 1, wherein the antibody or its antigen-binding fragment are selected from Fv, Fab, Fab', F (ab)₂、F (ab')₂, single-chain antibody, lack hinge area univalent antibody and whole antibody.

5. the antibody of claim 1, wherein at least one is applicable below：

（i）Wherein described antibody is with 10nM or lower KD combination people MASP-2；

（ii）Epitope in wherein described antibody binding MASP-2 CCP1 domains；

（iii）Wherein described antibody is with 10nM or lower IC ₅₀Suppress C3b depositions in external test in 1% human serum；

（iv）Wherein described antibody is with 30nM or lower IC ₅₀Suppress the C3b depositions in 90% human serum；And/or

（v）Wherein described antibody does not suppress classical pathway substantially.

6. antibody according to claim 1, wherein the antibody is selected from IgG2 molecules, IgG1 molecules and IgG4 molecules.

7. antibody according to claim 6, wherein the IgG4 molecules are mutated including S228P.

8. monoclonal antibody or its antigen-binding fragment with the people MASP-2 separation combined, it includes weight chain variable district, described Weight chain variable district includes any one in the amino acid sequence being listed below：SEQ ID NO：56 amino acid residue 1 to 120, SEQ ID NO：57 amino acid residue 1 to 120, SEQ ID NO：58 amino acid residue 1 to 120, SEQ ID NO： 60 amino acid residue 1 to 120 or SEQ ID NO：SEQ ID NO:61 amino acid residue 1 to 120；And light chain variable district, The light chain variable district includes any one in the amino acid sequence being listed below：SEQ ID NO：56 amino acid residue 146 to 250, SEQ ID NO：57 amino acid residue 146 to 250, SEQ ID NO：58 amino acid residue 146 to 250, SEQ ID NO：60 amino acid residue 146 to 250 or SEQ ID NO：61 amino acid residue 146 to 250.

9. nucleic acid molecules, it is encoded such as the MASP-2 antibody or the amino acid sequence of its fragment in claim 1 or 8.

10. expression cassette, it includes the nucleic acid molecules for encoding MASP-2 antibody of the invention according to claim 9.

11. cell, it includes the nucleic acid molecules of the MASP-2 antibody of the coding present invention according to claim 9 or claim 10 At least one of.

12. generating the method for the anti-MASP-2 antibody of separation, it is included in the nucleic acid molecules table for allowing to encode anti-MASP-2 antibody The cell of claim 11 is cultivated under conditions of reaching and the anti-MASP-2 antibody is separated.

13. composition, it includes the anti-MASP-2 antibody or its fragment and pharmaceutically acceptable excipient of claim 1 or 8.

14. composition according to claim 13, wherein the composition be formulated for intra-arterial, intravenous, encephalic, it is intramuscular, Suction, intranasal or subcutaneous administration.

15. the MASP-2 antibody as described in claim 1 or 8 is used to manufacture suppresses MASP-2 dependences in its subject is needed Complement activation medicine purposes.

16. product, it includes the anti-MASP- of human monoclonal for being suitable for the claim 1 or 8 that the treatment to people experimenter is applied The unit dose of 2 antibody, wherein the unit dose is 1mg-1000mg scopes.